Anti-MUC16 Antibodies, Antibody-Drug Conjugates, and Bispecific Antigen-Binding Molecules that Bind MUC16 and CD3, and Uses Thereof

ABSTRACT

Mucin 16 (MUC16) is highly expressed in ovarian cancer and expression on cancer cells is shown to protect tumor cells from the immune system. The present invention provides novel full-length human IgG antibodies that bind to human and MUC16 (monospecific antibodies). The present invention also provides novel bispecific antibodies (bsAbs) that bind to both MUC16 and CD3 and activate T cells via the CD3 complex in the presence of MUC16-expressing tumors. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human and monkey CD3, and a second antigen-binding molecule that specifically binds human and monkey MUC16. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of tumors expressing MUC16. The bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced MUC16-targeted immune response is desired and/or therapeutically beneficial. For example, the bispecific antibodies of the invention are useful for the treatment of various cancers, including ovarian cancer. The present invention also includes anti-MUC16 antibody drug conjugates which inhibit tumor growth in vivo. In some embodiments, the anti-MUC16 antibodies are useful in diagnostic methods for identifying the presence of MUC16 in tissue and/or plasma samples.

REFERENCE TO A SEQUENCE LISTING

This application is a continuation of U.S. application Ser. No.16/913,154, filed Jun. 26, 2020, which is a division of U.S. applicationSer. No. 15/713,574, filed Sep. 22, 2017, which claims the benefit under35 USC § 119(e) of US Provisional Application Nos. 62/399,249, filedSep. 23, 2016, and 62/558,711, filed Sep. 14, 2017, each of which isincorporated herein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference a computer readable SequenceListing in ST.26 format XML format, titled 10295US04-Sequence, createdon Sep. 26, 2022 and containing 2,379,761 bytes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for Mucin 16 (MUC16), and methodsof use thereof. The present invention also relates to bispecificantigen-binding molecules that bind MUC16 and CD3, and methods of usethereof. The present invention further relates to antibody-drugconjugates comprising an anti-MUC16 antibody or fragment thereof and atherapeutic agent (e.g., a cytotoxic agent).

BACKGROUND

Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen125, carbohydrate antigen 125, or CA-125, is a single transmembranedomain highly glycosylated integral membrane glycoprotein that is highlyexpressed in ovarian cancer. MUC16 consists of three major domains: anextracellular N-terminal domain, a large tandem repeat domaininterspersed with sea urchin sperm, enterokinase, and agrin (SEA)domains, and a carboxyl terminal domain that comprises a segment of thetransmembrane region and a short cytoplasmic tail. Proteolytic cleavageresults in shedding of much of the extracellular portion of MUC16 intothe bloodstream. MUC16 is overexpressed in cancers including ovariancancer, breast cancer, pancreatic cancer, non-small-cell lung cancer,intrahepatic cholangiocarcinoma-mass forming type, adenocarcinoma of theuterine cervix, and adenocarcinoma of the gastric tract, and in diseasesand conditions including inflammatory bowel disease, liver cirrhosis,cardiac failure, peritoneal infection, and abdominal surgery. (Haridas,D. et al., 2014, FASEB J., 28:4183-4199). Expression on cancer cells isshown to protect tumor cells from the immune system. (Felder, M. et al.,2014, Molecular Cancer, 13:129) Methods for treating ovarian cancerusing antibodies to MUC16 have been investigated. Oregovomab andabgovomab are anti-MUC16 antibodies which have had limited success.(Felder, supra, Das, S. and Batra, S. K. 2015, Cancer Res.75:4660-4674.)

CD3 is a homodimeric or heterodimeric antigen expressed on T cells inassociation with the T cell receptor complex (TCR) and is required for Tcell activation. Functional CD3 is formed from the dimeric associationof two of four different chains: epsilon, zeta, delta and gamma. The CD3dimeric arrangements include gamma/epsilon, delta/epsilon and zeta/zeta.Antibodies against CD3 have been shown to cluster CD3 on T cells,thereby causing T cell activation in a manner similar to the engagementof the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodieshave been proposed for therapeutic purposes involving the activation ofT cells. In addition, bispecific antibodies that are capable of bindingCD3 and a target antigen have been proposed for therapeutic usesinvolving targeting T cell immune responses to tissues and cellsexpressing the target antigen.

Antigen-binding molecules that target MUC16, including antibody-drugconjugates, as well as bispecific antigen-binding molecules that bindboth MUC16 and CD3 would be useful in therapeutic settings in whichspecific targeting and T cell-mediated killing of cells that expressMUC16 is desired.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides antibodies andantigen-binding fragments thereof that bind to human MUC16. Theantibodies according to this aspect of the invention are useful, interalia, for targeting cells expressing MUC16. The present invention alsoprovides bispecific antibodies and antigen-binding fragments thereofthat bind human MUC16 and human CD3. The bispecific antibodies accordingto this aspect of the invention are useful, inter alia, for targeting Tcells expressing CD3, and for stimulating T cell activation, e.g., undercircumstances where T cell-mediated killing of cells expressing MUC16 isbeneficial or desirable. For example, the bispecific antibodies candirect CD3-mediated T cell activation to specific MUC16-expressingcells, such as ovarian tumor cells.

Exemplary anti-MUC16 antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs) and light chainvariable regions (LCVRs), as well as heavy chain complementaritydetermining regions (HCDR1, HCDR2 and HCDR3), and light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) of theexemplary anti-MUC16 antibodies. Table 2 sets forth the sequenceidentifiers of the nucleic acid molecules encoding the HCVRs, LCVRs,HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-MUC16antibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-MUC16 antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is of SEQ ID NOs: 18/26 (e.g.,H1H8767P).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-MUC16 antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is of SEQ ID NOs:24/32 (e.g., H1H8767P).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-MUC16 antibodies listed in Table 1. In certainembodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of SEQ ID NOs:20-22-24-28-30-32 (e.g., H1H8767P).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRsHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary anti-MUC16antibodies listed in Table 1. For example, the present inventionincludes antibodies, or antigen-binding fragments thereof, comprisingthe HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences setcontained within an HCVR/LCVR amino acid sequence pair of SEQ ID NOs:18/26 (e.g., H1H8767P). Methods and techniques for identifying CDRswithin HCVR and LCVR amino acid sequences are well known in the art andcan be used to identify CDRs within the specified HCVR and/or LCVR aminoacid sequences disclosed herein. Exemplary conventions that can be usedto identify the boundaries of CDRs include, e.g., the Kabat definition,the Chothia definition, and the AbM definition. In general terms, theKabat definition is based on sequence variability, the Chothiadefinition is based on the location of the structural loop regions, andthe AbM definition is a compromise between the Kabat and Chothiaapproaches. See, e.g., Kabat, “Sequences of Proteins of ImmunologicalInterest,” National Institutes of Health, Bethesda, Md. (1991);Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al.,Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases arealso available for identifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-MUC16 antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs HCDR1-HCDR2-HCDR3),wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined byany of the exemplary anti-MUC16 antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs LCDR1-LCDR2-LCDR3),wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined byany of the exemplary anti-MUC16 antibodies listed in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-MUC16 antibody listed in Table1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-MUC16 antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-MUC16 antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds MUC16 and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an anti-MUC16 antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-MUC16 antibody. Additionalcombination therapies and co-formulations involving the anti-MUC16antibodies of the present invention are disclosed elsewhere herein.

In another aspect, the invention provides therapeutic methods fortargeting/killing tumor cells expressing MUC16 using an anti-MUC16antibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an anti-MUC16 antibody of the invention to asubject in need thereof. In some cases, the anti-MUC16 antibodies (orantigen-binding fragments thereof) can be used for treating cancer(e.g., ovarian cancer), or may be modified to be more cytotoxic bymethods, including but not limited to, modified Fc domains to increaseADCC (see e.g. Shield et al. (2002) JBC 277:26733), radioimmunotherapy,antibody-drug conjugates, or other methods for increasing the efficiencyof tumor ablation.

The present invention also includes the use of an anti-MUC16 antibody ofthe invention in the manufacture of a medicament for the treatment of adisease or disorder (e.g., cancer) related to or caused byMUC16-expressing cells. In one aspect, the invention relates to acompound comprising an anti-MUC16 antibody or antigen-binding fragment,or a MUC16×CD3 bispecific antibody, as disclosed herein, for use inmedicine. In one aspect, the invention relates to a compound comprisingan antibody-drug conjugate (ADC) as disclosed herein, for use inmedicine.

In yet another aspect, the invention provides monospecific anti-MUC16antibodies for diagnostic applications, such as, e.g., imaging reagents.

In yet another aspect, the invention provides therapeutic methods forstimulating T cell activation using an anti-CD3 antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody

In another aspect, the present invention provides an isolated antibodyor antigen-binding fragment thereof that binds human mucin 16 (MUC16)with a binding dissociation equilibrium constant (K_(D)) of less thanabout 53 nM as measured in a surface plasmon resonance assay at 25° C.In yet another aspect, the present invention provides an isolatedantibody or antigen-binding fragment thereof that binds human MUC16 witha dissociative half-life (t½) of greater than about 15 minutes asmeasured in a surface plasmon resonance assay at 25° C.

The invention further provides an antibody or antigen-binding fragmentthat competes for binding to human MUC16 with a reference antibodycomprising an HCVR/LCVR amino acid sequence pair as set forth inTable 1. In another aspect, the invention provides an antibody orantigen-binding fragment that competes for binding to human MUC16 with areference antibody comprising an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 2/10; 18/26; 34/42;50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170;178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266;274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and378/386.

The invention furthermore provides an antibody or antigen-bindingfragment, wherein the antibody or antigen-binding fragment thereof bindsto the same epitope on human MUC16 as a reference antibody comprising anHCVR/LCVR amino acid sequence pair as set forth in Table 1. In anotheraspect, the antibody or antigen-binding fragment binds to the sameepitope on human MUC16 as a reference antibody comprising an HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138;146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936;258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370;and 378/386.

The invention further provides an isolated antibody or antigen-bindingfragment thereof that binds human MUC16, wherein the antibody orantigen-binding fragment comprises: the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR) having an aminoacid sequence as set forth in Table 1; and the CDRs of a light chainvariable region (LCVR) having an amino acid sequence as set forth inTable 1. In another aspect, the isolated antibody or antigen-bindingfragment comprises the heavy and light chain CDRs of a HCVR/LCVR aminoacid sequence pair selected from the group consisting of: 2/10; 18/26;34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138; 146/154; 162/170;178/186; 194/394; 202/210; 218/226, 234/242; 250/1936; 258/266;274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and378/386. In yet another aspect, the isolated antibody or antigen-bindingfragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, selected from the group consisting of: SEQ ID NOs:4-6-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64;68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112;116-118-120-124-126-128; 132-134-136-140-142-144;148-150-152-156-158-160; 164-166-168-172-174-176;180-182-184-188-190-192; 196-198-200-396-398-400;204-206-208-212-214-216; 220-222-224-228-230-232;236-238-240-244-246-248; 252-254-256-1938-1940-1942;260-262-264-268-270-272; 276-278-280-1938-1940-1942;284-286-288-292-294-296; 300-302-304-308-310-312;316-318-320-324-326-328; 332-334-336-340-342-344;348-350-352-356-358-360; 364-366-368-372-374-376; and380-382-384-388-390-392.

In another aspect, the invention provides an isolated antibody orantigen-binding fragment thereof that binds human MUC16, wherein theantibody or antigen-binding fragment comprises: (a) a heavy chainvariable region (HCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, 178, 194, 202, 218, 234, 250, 258, 274, 282, 298, 314, 330,346, 362, and 378; and (b) a light chain variable region (LCVR) havingan amino acid sequence selected from the group consisting of SEQ ID NOs:10; 26; 42; 58, 74; 90; 106; 122; 138; 154; 170; 186; 210; 226, 242;266; 290; 306; 322; 338; 354; 370; 386; 1936 and 394. In a furtheraspect, the isolated antibody or antigen-binding fragment of claim 10,wherein the antibody or antigen-binding fragment comprises a HCVR/LCVRamino acid sequence pair selected from the group consisting of: SEQ IDNOs: 2/10; 18/26; 34/42; 50/58, 66/74; 82/90; 98/106; 114/122; 130/138;146/154; 162/170; 178/186; 194/394; 202/210; 218/226, 234/242; 250/1936;258/266; 274/1936; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370;and 378/386.

The invention further provides an isolated antibody or antigen-bindingfragment thereof that binds human MUC16 within an epitope ranging fromresidue 428 to residue 481 of SEQ ID NO: 1902. In some cases, theisolated antibody or antigen-binding fragment interacts with amino acidresidues 428-434, 429-434, 453-467, 459-467, 460-467 and/or 474-481 ofSEQ ID NO: 1902. In some embodiments, the antibody or antigen-bindingfragment interacts with amino acid residues 428-434, 429-434, 453-467,459-467, 460-467 and 474-481 of SEQ ID NO: 1902. The invention furtherprovides an isolated antibody or antigen-binding fragment thereof thatbinds human MUC16 within an epitope ranging from residue 126 to residue138 of SEQ ID NO: 1902. In some cases, the isolated antibody orantigen-binding fragment interacts with amino acid residues 126-131,127-131 and/or 132-138 of SEQ ID NO: 1902. In some embodiments, theantibody or antigen-binding fragment interacts with amino acid residues126-131, 127-131 and 132-138 of SEQ ID NO: 1902. The invention furtherprovides an isolated antibody or antigen-binding fragment thereof thatbinds human MUC16 within an epitope ranging from residue 357 to residue369 of SEQ ID NO: 1902. In some cases, the isolated antibody orantigen-binding fragment interacts with amino acid residues 357-369,358-366, 358-369 and/or 361-369 of SEQ ID NO: 1902. In some embodiments,the antibody or antigen-binding fragment interacts with amino acidresidues 357-369, 358-366, 358-369 and 361-369 of SEQ ID NO: 1902. Theinvention further provides an isolated antibody or antigen-bindingfragment thereof that binds human MUC16 within one or more of the fivemembrane-proximal SEA domains of human MUC 16 (SEQ ID NO: 1899). Thefive membrane-proximal SEA domains correspond to residues 13791-14451 ofSEQ ID NO: 1899. In some cases, the antibody or antigen-binding fragmentbinds with a K_(D) of less than about 60 nM as measured in a surfaceplasmon resonance assay at 25° C. In some embodiments, the antibody orantigen-binding fragment binds within residues 14237 to 14290 of SEQ IDNO: 1899. In one embodiment, the antibody or antigen-binding fragmentcomprises CDRs of a HCVR/LCVR pair comprising the amino acid sequencesof SEQ ID NO: 18/26. In some embodiments, the antibody orantigen-binding fragment binds within residues 13935 to 13947 of SEQ IDNO: 1899. In one embodiment, the antibody or antigen-binding fragmentcomprises CDRs of a HCVR/LCVR pair comprising the amino acid sequencesof SEQ ID NO: 82/858. In some embodiments, the antibody orantigen-binding fragment binds within residues 14165 to 14178 of SEQ IDNO: 1899. In one embodiment, the antibody or antigen-binding fragmentcomprises CDRs of a HCVR/LCVR pair comprising the amino acid sequencesof SEQ ID NO: 98/170.

In one aspect, the invention provides antibodies or antigen-bindingfragments thereof that bind to one of more of the SEA domains of MUC16.In various embodiments, the anti-MUC16 antibodies or antigen-bindingfragments bind to any one of more of SEA1, SEA2, SEA3, SEA4, SEA5, SEA6,SEA7, SEA8, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15 or SEA16. Inone embodiment, the anti-MUC16 antibody or fragment binds within SEA1(residues 12074 to 12229 of SEQ ID NO: 1899). In one embodiment, theanti-MUC16 antibody or fragment binds within SEA2 (residues 12230 to12387 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody orfragment binds within SEA3 (residues 12388 to 12543 of SEQ ID NO: 1899).In one embodiment, the anti-MUC16 antibody or fragment binds within SEA4(residues 12544 to 12698 of SEQ ID NO: 1899). In one embodiment, theanti-MUC16 antibody or fragment binds within SEA5 (residues 12699 to12854 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody orfragment binds within SEA6 (residues 12855 to 13010 of SEQ ID NO: 1899).In one embodiment, the anti-MUC16 antibody or fragment binds within SEA7(residues 13011 to 13166 of SEQ ID NO: 1899). In one embodiment, theanti-MUC16 antibody or fragment binds within SEA8 (residues 13167 to13323 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody orfragment binds within SEA9 (residues 13324 to 13478 of SEQ ID NO: 1899).In one embodiment, the anti-MUC16 antibody or fragment binds withinSEA10 (residues 13479 to 13634 of SEQ ID NO: 1899). In one embodiment,the anti-MUC16 antibody or fragment binds within SEA11 (residues 13635to 13790 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibodyor fragment binds within SEA12 (residues 13791 to 13923 of SEQ ID NO:1899). In one embodiment, the anti-MUC16 antibody or fragment bindswithin SEA13 (residues 13924 to 14074 of SEQ ID NO: 1899). In oneembodiment, the anti-MUC16 antibody or fragment binds within SEA14(residues 14075 to 14227 of SEQ ID NO: 1899). In one embodiment, theanti-MUC16 antibody or fragment binds within SEA15 (residues 14228 to14320 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody orfragment binds within SEA16 (residues 14321 to 14464 of SEQ ID NO:1899).

According to another aspect, the present invention providesantibody-drug conjugates comprising an anti-MUC16 antibody orantigen-binding fragment thereof and a therapeutic agent (e.g., acytotoxic agent). In some embodiments, the antibody or antigen-bindingfragment and the cytotoxic agent are covalently attached via a linker,as discussed herein. In various embodiments, the anti-MUC16 antibody orantigen-binding fragment can be any of the anti-MUC16 antibodies orfragments described herein.

In some embodiments, the cytotoxic agent is selected from an auristatin,a maytansinoid, a tubulysin, a tomaymycin derivative, or a dolastatinderivative. In some cases, the cytotoxic agent is an auristatin selectedfrom MMAE or MMAF, or a maytansinoid selected from DM1 or DM4. In someembodiments, the cytotoxic agent is a maytansinoid having the structureof Formula (I) or Formula (II), as discussed herein.

In some embodiments, the cytotoxic agent is a maytansinoid having thestructure:

In some embodiments, the cytotoxic agent is a maytansinoid having thestructure:

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16antibody or fragment thereof, and

wherein

is a bond to the anti-MUC16 antibody or fragment thereof.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16antibody or fragment thereof, and

wherein

is a bond to the anti-MUC16 antibody or fragment thereof.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16antibody or fragment thereof, and

wherein

is a bond to the anti-MUC16 antibody or fragment thereof.

In some embodiments, the bond contacts the antibody or fragment thereofvia a sulfur constituent of a cysteine residue.

In some embodiments, the antibody-drug conjugate comprises an anti-MUC16antibody or fragment thereof, and

or

a mixture thereof,

wherein

is a bond to the anti-MUC16 antibody or fragment thereof.

In some embodiments, the bond contacts the antibody or fragment thereofvia a nitrogen constituent of a lysine residue.

In any of the various embodiments of the antibody-drug conjugatesdiscussed above or herein, the antibody-drug conjugate can comprise from1 to 4 cytotoxic agents per anti-MUC16 antibody or fragment thereof.

According to another aspect, the present invention provides bispecificantigen-binding molecules (e.g., antibodies) that bind MUC16 and CD3.Such bispecific antigen-binding molecules are also referred to herein as“anti-MUC16/anti-CD3 bispecific molecules,” “anti-CD3/anti-MUC16bispecific molecules,” or “MUC16×CD3 bsAbs.” The anti-MUC16 portion ofthe anti-MUC16/anti-CD3 bispecific molecule is useful for targetingcells (e.g., tumor cells) that express MUC16 (e.g., ovarian tumors), andthe anti-CD3 portion of the bispecific molecule is useful for activatingT-cells. The simultaneous binding of MUC16 on a tumor cell and CD3 on aT-cell facilitates directed killing (cell lysis) of the targeted tumorcell by the activated T-cell. The anti-MUC16/anti-CD3 bispecificmolecules of the invention are therefore useful, inter alia, fortreating diseases and disorders related to or caused by MUC16-expressingtumors (e.g., ovarian cancers).

The bispecific antigen-binding molecules according to this aspect of thepresent invention comprise a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds MUC16. The present invention includesanti-MUC16/anti-CD3 bispecific molecules (e.g., bispecific antibodies)wherein each antigen-binding domain comprises a heavy chain variableregion (HCVR) paired with a light chain variable region (LCVR). Incertain exemplary embodiments of the invention, the anti-CD3antigen-binding domain and the anti-MUC16 antigen binding domain eachcomprise different, distinct HCVRs paired with a common LCVR. Forexample, as illustrated in Example 3 herein, bispecific antibodies wereconstructed comprising a first antigen-binding domain that specificallybinds CD3, wherein the first antigen-binding domain comprises an HCVRderived from an anti-CD3 antibody paired with an LCVR derived from ananti-MUC16 antibody (e.g., the same LCVR that is included in theanti-MUC16 antigen-binding domain); and a second antigen-binding domainthat specifically binds MUC16, wherein the second antigen-binding domaincomprises an HCVR/LCVR derived from an anti-MUC16 antibody. In otherwords, in the exemplary molecules disclosed herein, the pairing of anHCVR from an anti-CD3 antibody with an LCVR from an anti-MUC16 antibodycreates an antigen-binding domain that specifically binds CD3 (but doesnot bind MUC16). In such embodiments, the first and secondantigen-binding domains comprise distinct anti-CD3 and anti-MUC16 HCVRsbut share a common anti-MUC16 LCVR. In other embodiments, the bispecificantigen-binding molecules comprise distinct anti-CD3 and anti-MUC16HCVRs, but share a common LCVR. The amino acid sequence of this LCVR isshown, e.g., in SEQ ID NO:1890, and the amino acid sequences of thecorresponding CDRs LCDR1-LCDR2-LCDR3) are shown in SEQ ID NOs:1892, 1894and 1896, respectively. Genetically modified mice can be used to producefully human bispecific antigen-binding molecules comprising twodifferent heavy chains that associate with an identical light chain thatcomprises a variable domain derived from one of two different humanlight chain variable region gene segments. Alternatively, variable heavychains may be paired with one common light chain and expressedrecombinantly in host cells. As such, the antibodies of the inventioncan comprise immunoglobulin heavy chains associated with a singlerearranged light chain. In some embodiments, the light chain comprises avariable domain derived from a human Vκ1-39 gene segment or a Vκ3-20gene segment. In other embodiments, the light chain comprises a variabledomain derived from a human Vκ1-39 gene segment rearranged with a humanJκ5 or a human Jκ1 gene segment.

The present invention provides anti-CD3/anti-MUC16 bispecific molecules,wherein the first antigen-binding domain that specifically binds CD3comprises any of the HCVR amino acid sequences, any of the LCVR aminoacid sequences, any of the HCVR/LCVR amino acid sequence pairs, any ofthe heavy chain CDR1-CDR2-CDR3 amino acid sequences, or any of the lightchain CDR1-CDR2-CDR3 amino acid sequences as set forth in US publication2014/0088295 published Mar. 27, 2014 and PCT/US2016/044732 filed Jul.29, 2016.

In addition, the present invention provides anti-CD3/anti-MUC16bispecific molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises any of the HCVR amino acid sequences asset forth in Tables 16, 18, and 22 herein. The first antigen-bindingdomain that specifically binds CD3 may also comprise any of the LCVRamino acid sequences as set forth in Tables 1, 16, 19, and 23 herein.According to certain embodiments, the first antigen-binding domain thatspecifically binds CD3 comprises any of the HCVR/LCVR amino acidsequence pairs as set forth in Tables 16, 18, 19, 22, and 23 herein. Thepresent invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises any of the heavy chain CDR1-CDR2-CDR3 amino acidsequences as set forth in Tables 16, 18, and 22 herein, and/or any ofthe light chain CDR1-CDR2-CDR3 amino acid sequences as set forth inTables 1, 16, 19, and 23 herein.

According to certain embodiments, the present invention providesanti-CD3/anti-MUC16 bispecific molecules, wherein the firstantigen-binding domain that specifically binds CD3 comprises a heavychain variable region (HCVR) having an amino acid sequence as set forthin Tables 16, 18, and 22 herein or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a light chain variable region (LCVR) having an aminoacid sequence as set forth in Tables 1, 6, 19, and 23 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequence pairas set forth in Tables 16, 18, 19, 22, and 23 herein.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the first antigen-binding domain that specificallybinds CD3 comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence as set forth in Tables 16, 18, and 22 herein, or asubstantially similar sequence thereto having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR3 (LCDR3) domain having an amino acid sequence as set forth in Tables1, 16, 19, and 23 herein, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In certain embodiments, the first antigen-binding domain thatspecifically binds CD3 comprises a HCDR3/LCDR3 amino acid sequence pairas set forth in Tables 16, 18, 19, 22, and 23 herein.

The present invention also provides anti-CD3/anti-MUC16 bispecificantigen-binding molecules, wherein the first antigen-binding domain thatspecifically binds CD3 comprises a heavy chain CDR1 (HCDR1) domainhaving an amino acid as set forth in Tables 16, 18, and 22 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2(HCDR2) domain having an amino acid as set forth in Tables 16, 18, and22, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a heavy chainCDR3 (HCDR3) domain having an amino acid as set forth in Tables 16, 18,and 22, or a substantially similar sequence thereof having at least 90%,at least 95%, at least 98% or at least 99% sequence identity; a lightchain CDR1 (LCDR1) domain having an amino acid sequence as set forth inTables 1, 16, 19, and 23 herein, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a light chain CDR2 (LCDR2) domain having an aminoacid sequence as set forth in Tables 1, 16, 19, and 23 herein, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity, and a light chainCDR3 (LCDR3) domain having an amino acid sequence as set forth in Tables1, 16, 19, and 23 herein, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Certain non-limiting, exemplary anti-CD3/anti-MUC16 bispecificantigen-binding molecules of the invention include a firstantigen-binding domain that specifically binds CD3 comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences as set forth in Tables 16, 18, 19, 22, and 23herein.

The present invention further provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) comprising an amino acid sequence as set forth in Table 16, Table18, or Table 22 and light chain complementarity determining regions(LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR)comprising an amino acid sequence as set forth in Table 1, Table 16,Table 19 or Table 23.

In another aspect, the invention provides a bispecific antigen-bindingmolecule wherein the first antigen-binding domain that specificallybinds human CD3 comprises heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region(HCVR) selected from the group consisting of SEQ ID NOs: 1730, 1762,1778, 1786, and 1866, and light chain complementarity determiningregions (LCDR1, LCDR2 and LCDR3) from a light chain variable region(LCVR) comprising an amino acid sequence of SEQ ID NO: 26.

The invention further provides a bispecific antigen-binding molecule,wherein the first antigen-binding domain that specifically binds humanCD3 comprises three heavy chain complementarity determining regions(A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementaritydetermining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), wherein A1-HCDR1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs:1734,1766, 1782, 1790, and 1870; A1-HCDR3 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs:1736, 1768, 1784, 1792,and 1872; A1-LCDR1 comprises an amino acid sequence of SEQ ID NO:28;A1-LCDR2 comprises an amino acid sequence of SEQ ID NO:30; and A1-LCDR3comprises an amino acid sequence of SEQ ID NO:32.

In a further aspect, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises the heavy and light chain CDRs of a HCVR/LCVRamino acid sequence pair selected from the group consisting of: SEQ IDNOs: 1730/26, 1762/26, 1778/26, 1786/26, and 1866/26

In another aspect, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain that specificallybinds human CD3 comprises three heavy chain complementarity determiningregions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chaincomplementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3),and wherein the second antigen-binding domain that specifically bindshuman MUC16 comprises three heavy chain complementarity determiningregions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chaincomplementarity determining regions (A2-LCDR1, A2-LCDR2 and A2-LCDR3);wherein A1-HCDR1 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1732, 1764, 1780, 1788, and 1868;A1-HCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1734, 1766, 1782, 1790, and 1870; A1-HCDR3comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1736, 1768, 1784, 1792, and 1872; A1-LCDR1 comprises anamino acid sequence of SEQ ID NO:28; A1-LCDR2 comprises an amino acidsequence of SEQ ID NO:30; and A1-LCDR3 comprises an amino acid sequenceof SEQ ID NO:32; and wherein A2-HCDR1 comprises an amino acid sequenceof SEQ ID NO:20; A2-HCDR2 comprises an amino acid sequence of SEQ IDNO:22; A2-HCDR3 comprises an amino acid sequence of SEQ ID NO:24;A2-LCDR1 comprises an amino acid sequence of SEQ ID NO:28; A2-LCDR2comprises an amino acid sequence of SEQ ID NO:30; and A2-LCDR3 comprisesan amino acid sequence of SEQ ID NO:32.

Certain non-limiting, exemplary anti-CD3/anti-MUC16 bispecificantigen-binding molecules of the invention include a firstantigen-binding domain that specifically binds CD3 comprising a heavychain comprising variable domain framework regions having an amino acidsequence selected from FR1 (SEQ ID NO: 1903), FR2 (SEQ ID NO: 1904), FR3(SEQ ID NO: 1905), and FR4 (SEQ ID NO: 1906).

In more embodiments, exemplary anti-CD3/anti-MUC16 bispecificantigen-binding molecules of the invention include a bispecificantigen-binding molecule wherein the first antigen-binding domain thatspecifically binds human CD3 comprises a HCVR comprisingHCDR1-HCDR2-HCDR3 having the amino acid sequences of SEQ ID NOs:1907-1908-1909.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the second antigen-binding domain that specificallybinds MUC16 comprises a heavy chain variable region (HCVR) having theamino acid sequence selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 202, 218, 234,250, 258, 274, 282, 298, 314, 330, 346, 362, and 378, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the second antigen-binding domain that specificallybinds MUC16 comprises a light chain variable region (LCVR) having theamino acid sequence selected from the group consisting of SEQ ID NOs:10;26; 42; 58, 74; 90; 106; 122; 138; 154; 170; 186; 210; 226, 242; 266;290; 306; 322; 338; 354; 370; 386; 1936; and 394, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the second antigen-binding domain that specificallybinds MUC16 comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequencepair selected from the group consisting of SEQ ID NOs:18/26.

The present invention also provides anti-CD3/anti-MUC16 bispecificmolecules, wherein the second antigen-binding domain that specificallybinds MUC16 comprises a heavy chain CDR3 (HCDR3) domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs:8, 24,40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 208, 224, 240, 256,264, 280, 288, 304, 320, 336, 352, 368, and 384, or a substantiallysimilar sequence thereto having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NOs:16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 216, 232,248, 272, 296, 312, 328, 344, 360, 376, 392, 400, and 1942, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the second antigen-binding domain thatspecifically binds MUC16 comprises a HCDR3/LCDR3 amino acid sequencepair selected from the group consisting of SEQ ID NOs: 24/32.

The present invention also provides anti-CD3/anti-MUC16 bispecificantigen-binding molecules, wherein the second antigen-binding domainthat specifically binds MUC16 comprises a heavy chain CDR1 (HCDR1)domain having an amino acid sequence selected from the group consistingof SEQ ID NOs:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196,204, 220, 236, 252, 260, 276, 284, 300, 316, 332, 348, 364, and 380, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2(HCDR2) domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs:6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166,182, 198, 206, 222, 238, 254, 262, 278, 286, 302, 318, 334, 350, 366,and 382, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; aheavy chain CDR3 (HCDR3) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:8, 24, 40, 56, 72, 88, 104, 120,136, 152, 168, 184, 200, 208, 224, 240, 256, 264, 280, 304, 320, 336,352, 368, and 384, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a light chain CDR1 (LCDR1) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:12, 28, 44, 60, 76, 92, 108,124, 140, 156, 172, 188, 396, 212, 228, 244, 396, 268, 396, 292, 308,324, 340, 356, 372, 1938, and 388, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; and a light chain CDR2 (LCDR2) domain having an aminoacid sequence selected from the group consisting of SEQ ID NOs:14, 30,46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 398, 214, 230, 246, 398,270, 398, 294, 310, 326, 342, 358, 374, 1940, and 390, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR3 (LCDR3) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs:16, 32, 48, 64, 80, 96, 112, 128, 144,160, 176, 192, 400, 216, 232, 248, 400, 272, 400, 296, 312, 328, 344,360, 376, 1942, and 392, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Certain non-limiting, exemplary anti-CD3/anti-MUC16 bispecificantigen-binding molecules of the invention include a secondantigen-binding domain that specifically binds MUC16 comprisingHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences selected from the group consisting of: SEQ ID NOs:20-22-24-28-30-32.

In a related embodiment, the invention includes anti-CD3/anti-MUC16bispecific antigen-binding molecules wherein the second antigen-bindingdomain that specifically binds MUC16 comprises the heavy and light chainCDR domains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NOs:18/26.

In one embodiment, the invention provides an anti-CD3/anti-MUC16bispecific antibody, comprising an anti-MUC16 binding arm that comprisesa heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 anda light chain comprising the amino acid sequence of SEQ ID NO: 1960, andan anti-CD3 binding arm that comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 1961 and a light chain comprising theamino acid sequence of SEQ ID NO: 1960. In another embodiment, theinvention provides an anti-CD3/anti-MUC16 bispecific antibody,comprising an anti-MUC16 binding arm that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 1959 and a light chaincomprising the amino acid sequence of SEQ ID NO: 1960, and an anti-CD3binding arm that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 1962 and a light chain comprising the amino acidsequence of SEQ ID NO: 1960.

In another aspect, the invention provides a bispecific antigen-bindingmolecule comprising a first antigen-binding domain that binds human CD3and a second antigen-binding domain that binds human MUC16, wherein thesecond antigen-binding domain is derived from the antibody orantigen-binding fragment of any one of the anti-MUC16 antibodies of theinvention. In a further aspect, the invention provides a bispecificantigen-binding molecule comprising a first antigen-binding domain thatspecifically binds human CD3, and a second antigen-binding domain thatspecifically binds human MUC16.

The invention further provides a bispecific antigen-binding moleculewhich binds human cells expressing human CD3 and cynomolgus monkey cellsexpressing cynomolgus CD3. In another aspect, the bispecificantigen-binding molecule binds human cells expressing human MUC16.

In another aspect the invention provides a bispecific antigen-bindingmolecule which inhibits tumor growth in immunocompromised mice bearinghuman ovarian cancer xenografts. The invention further provides abispecific antigen-binding molecule which suppresses tumor growth ofestablished tumors in immunocompromised mice bearing human ovariancancer xenografts.

In another aspect the invention provides a bispecific antigen-bindingmolecule comprising i) a first antigen-binding domain that specificallybinds an effector cell with an EC₅₀ value of greater than about 4 nMand, and ii) a second antigen-binding domain that specifically binds atarget human ovarian tumor cell with an EC₅₀ value of less than 3 nM,wherein such EC₅₀ binding value is measured in an in vitro FACS bindingassay.

In one embodiment, the bispecific antigen-binding molecule can include asecond antigen-binding domain that specifically binds the target ovariantumor cell with an EC₅₀ value of less than about 2 nM. In some cases,the first antigen-binding domain specifically binds each of human CD3and cynomolgus CD3 with an EC₅₀ value of greater than about 40 nM,greater than about 100 nM, greater than about 200 nM, greater than about300 nM, greater than about 400 nM, greater than about 500 nM, or greaterthan about 1 μM. In some cases, the first antigen-binding domainspecifically binds each of human CD3 and cynomolgus CD3 with weak or nomeasurable binding or binding affinity.

In some embodiments, the antigen-binding molecule induces Tcell-mediated tumor cell killing with an EC₅₀ value of less than about31 pM, as measured in an in vitro T cell-mediated tumor cell killingassay, for example, where the tumor cells are OVCAR3 cells.

In some applications, the first antigen-binding domain binds human CD3with an K_(D) value of greater than about 11 nM, as measured in an invitro surface plasmon resonance binding assay. In some instances, thefirst antigen-binding domain binds each of human CD3 and cynomolgus CD3with an K_(D) value of greater than about 15 nM, greater than about 30nM, greater than about 60 nM, greater than about 120 nM, greater thanabout 300 nM, or greater than about 500 nM as measured in an in vitrosurface plasmon resonance binding assay.

In certain embodiments, anti-CD3 antibodies of the invention,antigen-binding fragments and bispecific antibodies thereof were made byreplacing amino acid residues of a parental in a stepwise manner basedon differences between the germline sequence and the parental antibodysequence.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the second antigen-binding domain competes for bindingto human MUC16 with a reference antigen-binding protein comprising threeheavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2 andA2-HCDR3) and three light chain complementarity determining regions(A2-LCDR1, A2-LCDR2 and A2-LCDR3), wherein A2-HCDR1 comprises an aminoacid sequence of SEQ ID NO: 20; A2-HCDR2 comprises an amino acidsequence of SEQ ID NO: 22; A2-HCDR3 comprises an amino acid sequence ofSEQ ID NO: 24; A2-LCDR1 comprises an amino acid sequence of SEQ ID NO:28; A2-LCDR2 comprises an amino acid sequence of SEQ ID NO: 30; andA2-LCDR3 comprises an amino acid sequence of SEQ ID NO:32. In someembodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the second antigen-binding domain competes for bindingto human MUC16 with a reference antigen-binding protein comprising aheavy chain variable region (HCVR) comprising an amino acid sequence ofSEQ ID NO: 18, and a light chain variable region (LCVR) comprising anamino acid sequence SEQ ID NO: 26.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain competes for bindingto human CD3 with a reference antigen-binding protein comprising threeheavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 andA1-HCDR3) and three light chain complementarity determining regions(A1-LCDR1, A1-LCDR2 and A1-LCDR3), A1-HCDR1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 1732, 1764,1780, 1788, and 1868; A1-HCDR2 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790, and1870; A1-HCDR3 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1736, 1768, 1784, 1792, and 1872; A1-LCDR1comprises an amino acid sequence SEQ ID NO: 28; A1-LCDR2 comprises anamino acid sequence of SEQ ID NO: 30; and A1-LCDR3 comprises an aminoacid sequence of SEQ ID NO: 32. In some embodiments, the inventionprovides a bispecific antigen-binding molecule, wherein the firstantigen-binding domain competes for binding to human CD3 with areference antigen-binding protein comprising a heavy chain variableregion (HCVR) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1730, 1762, 1778, 1786, and 1866, and a lightchain variable region (LCVR) comprising an amino acid sequence of SEQ IDNO:26.

In some embodiments, the invention provides a bispecific antigen-bindingmolecule, wherein the first antigen-binding domain competes for bindingto human CD3 with a reference antigen-binding protein comprising a heavychain variable region (HCVR) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786, and1866, and a light chain variable region (LCVR) comprising an amino acidsequence of SEQ ID NO:26; and wherein the second antigen-binding domaincompetes for binding to human MUC16 with a reference antigen-bindingprotein comprising a heavy chain variable region (HCVR) comprising anamino acid sequence of SEQ ID NO:18, and a light chain variable region(LCVR) comprising an amino acid sequence of SEQ ID NO: 26.

In one aspect, the invention provides a pharmaceutical compositioncomprising an anti-MUC16 antigen-binding molecule or anti-MUC16/anti-CD3bispecific antigen-binding molecule and a pharmaceutically acceptablecarrier or diluent. The invention further provides a method for treatinga cancer in a subject, the method comprising administering to thesubject the pharmaceutical composition comprising an anti-MUC16antigen-binding molecule or anti-MUC16/anti-CD3 bispecificantigen-binding molecule and a pharmaceutically acceptable carrier ordiluent. In some embodiments, the cancer is selected from the groupconsisting of cancers including ovarian cancer, breast cancer,pancreatic cancer, non-small-cell lung cancer, intrahepaticcholangiocarcinoma-mass forming type, adenocarcinoma of the uterinecervix, and adenocarcinoma of the gastric tract. In some cases, thecancer is ovarian cancer.

In another aspect, the present invention provides nucleic acid moleculesencoding any of the HCVR, LCVR or CDR sequences of theanti-CD3/anti-MUC16 bispecific antigen-binding molecules disclosedherein, including nucleic acid molecules comprising the polynucleotidesequences as set forth in Tables 2, 17, 20, 21, 23, and 25 herein, aswell as nucleic acid molecules comprising two or more of thepolynucleotide sequences as set forth in Tables 2, 17, 20, 21, 23, and25 in any functional combination or arrangement thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

The present invention includes anti-CD3/anti-MUC16 bispecificantigen-binding molecules wherein any of the aforementionedantigen-binding domains that specifically bind CD3 are combined,connected or otherwise associated with any of the aforementionedantigen-binding domains that specifically bind MUC16 to form abispecific antigen-binding molecule that binds CD3 and MUC16.

The present invention includes anti-CD3/anti-MUC16 bispecificantigen-binding molecules having a modified glycosylation pattern. Insome applications, modification to remove undesirable glycosylationsites may be useful, or an antibody lacking a fucose moiety present onthe oligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, modification of galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising an anti-CD3/anti-MUC16 bispecific antigen-binding molecule asdisclosed herein and a pharmaceutically acceptable carrier. In a relatedaspect, the invention features a composition which is a combination ofan anti-CD3/anti-MUC16 bispecific antigen-binding molecule and a secondtherapeutic agent. In one embodiment, the second therapeutic agent isany agent that is advantageously combined with an anti-CD3/anti-MUC16bispecific antigen-binding molecule. Exemplary agents that may beadvantageously combined with an anti-CD3/anti-MUC16 bispecificantigen-binding molecule are discussed in detail elsewhere herein.

In yet another aspect, the invention provides therapeutic methods fortargeting/killing tumor cells expressing MUC16 using ananti-CD3/anti-MUC16 bispecific antigen-binding molecule of theinvention, wherein the therapeutic methods comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising an anti-CD3/anti-MUC16 bispecific antigen-binding molecule ofthe invention to a subject in need thereof.

The present invention also includes the use of an anti-CD3/anti-MUC16bispecific antigen-binding molecule of the invention in the manufactureof a medicament for the treatment of a disease or disorder related to orcaused by MUC16-expressing cells.

In another aspect, the present invention provides a method of detectingMUC16 in a biological sample, comprising: obtaining a biological samplefrom a subject, and detecting whether MUC16 is present in the biologicalsample by contacting the biological sample with an anti-MUC16 antibodyor antigen-binding fragment thereof and detecting binding between MUC16and the anti-MUC16 antibody or antigen-binding fragment. In some cases,the biological sample is a tissue or fluid sample selected from plasma,serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas,stomach, small or large intestine, gall bladder, breast, lung, kidney,salivary, and lacrimal glands, or any epithelioid malignancy thereof. Insome embodiments, the antibody or antigen-binding fragment binds humanMUC16 within one or more of five membrane-proximal SEA domains of humanMUC16 corresponding to residues 13791-14451 of SEQ ID NO: 1899. In someembodiments, the antibody or antigen-binding fragment binds human MUC16within residues 13810-14451 of SEQ ID NO: 1899. In some embodiments, theantibody or antigen-binding fragment binds to any one of more of SEA1,SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEAR, SEA9, SEA10, SEA11, SEA12,SEA13, SEA14, SEA15 or SEA16 of human MUC16.

In another aspect, the invention provides a method of detecting MUC16 ina patient, comprising: obtaining a tissue sample from the patient; anddetecting whether MUC16 is present in the tissue sample by contactingthe tissue sample with an anti-MUC16 antibody and detecting bindingbetween MUC16 and the anti-MUC16 antibody. In some cases, the methodfurther comprises diagnosing the patient with a cancer when the presenceof MUC16 in the tissue sample is detected. In one embodiment, the tissuesample is ovarian tissue. In some cases, the anti-MUC16 antibody isspecific for an epitope within residues 12783-13467 of SEQ ID NO: 1899.In one embodiment, the anti-MUC16 antibody comprises CDRs of a HCVR/LCVRpair comprising the amino acid sequences of SEQ ID NO: 202/210. In somecases, the anti-MUC16 antibody is specific for an epitope withinresidues 13810-14451 of SEQ ID NO: 1899. In one embodiment, theanti-MUC16 antibody comprises CDRs of a HCVR/LCVR amino acid sequencepair selected from the group consisting of SEQ ID NO: 250/1936, 258/266,314/322 and 1944/1952.

In another aspect, the invention provides a method of detecting MUC16 ina patient, comprising: obtaining a plasma sample from the patient; anddetecting whether MUC16 is present in the plasma sample by contactingthe plasma sample with an anti-MUC16 antibody comprising CDRs of aHCVR/LCVR pair comprising the amino acid sequences of SEQ ID NO:202/210, and detecting binding between MUC16 and the anti-MUC16antibody. In some cases, the method further comprises diagnosing thepatient with a cancer when the presence of MUC16 in the plasma sample isdetected. In some embodiments, the method further comprisesadministering an effective amount of an anti-CD3×MUC16 bispecificantibody to the diagnosed patient. In some embodiments, the methodfurther comprises administering an effective amount of an ADC comprisingan anti-MUC16 antibody or antigen-binding fragment thereof and acytotoxic agent to the diagnosed patient.

In another aspect, the invention provides an isolated antibody orantigen-binding fragment thereof that binds MUC16, wherein the antibodyor antigen-binding fragment comprises CDRs of a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NO: 202/210,250/1936, 258/266, 314/322, 82/858, 98/170, and 1944/1952. In someembodiments, the antibody or antigen-binding fragment thereof comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected fromthe group consisting of SEQ ID NOs: 204-206-208-212-214-216;252-254-256-1938-1940-1942; 260-262-264-268-270-272;316-318-320-324-326-328; 84-86-88-1892-1894-1896;100-102-104-172-174-176; and 1946-1948-1950-1954-1956-1958. In someembodiments, the antibody or antigen-binding fragment comprises aHCVR/LCVR amino acid sequence pair selected from the group consisting of202/210, 250/1936, 258/266, 314/322, 82/858, 98/170, and 1944/1952.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2, and 3 illustrate pharmacokinetic profiles of anti-MUC16×CD3bispecific antibodies in wild-type mice (FIG. 1 ), humanized CD3 mice(FIG. 2 ) or humanized MUC16×CD3 mice (FIG. 3 ).

FIG. 4 shows the results of the OVCAR-3 model study 1 (Avg Radiance[p/s/cm²/sr] at Day 6). All groups had similar tumor burden as assessedby BLI before dosing started. Data shown is tumor burden as assessed byBLI on Day 6 post tumor implantation. Statistical significance wasdetermined using unpaired nonparametric Mann-Whitney t-tests. There wasno significant difference in tumor burden between groups.

FIG. 5 shows the results of the OVCAR-3 model study 1 (Avg Radiance[p/s/cm2²/sr] at Day 20). BSMUC16/CD3-001 significantly reduces tumorburden at 0.1 and 0.5 mg/kg. NSG mice engrafted with human T cells wereimplanted with human OVCAR-3/Luc cells. Treatment began 6 days posttumor implantation. Mice were treated on Days 6, 10, 13, 16 and 21 with0.01, 0.1, or 0.5 mg/kg BSMUC16/CD3-001 administered IP or treated witha CD3-binding control or non-binding control (0.5 mg/kg IP). Data shownis tumor burden as assessed by BLI on Day 20 post tumor implantation.Statistical significance was determined using unpaired nonparametricMann-Whitney t-tests. Treatment with BSMUC16/CD3-001 was compared to thenon-binding control (** p<0.01 for 0.5 mg/kg, #p<0.05 for 0.1 mg/kgBSMUC16/CD3-001).

FIG. 6 shows the results of the OVCAR-3 model study 1 (Fold change inBLI-evident tumors between D6 and D20). BSMUC16/CD3-001 significantlyreduces fold change in tumor burden at 0.01, 0.1, and 0.5 mg/kg. NSGmice engrafted with human T cells were implanted with human OVCAR-3/Luccells. Mice were treated on Days 6, 10, 13, 16 and 21 with 0.01, 0.1 or0.5 mg/kg BSMUC16/CD3-001 administered IP or treated with a CD3-bindingcontrol or non-binding control (0.5 mg/kg IP). Data shown is fold changein tumor burden from first measurement (taken before treatment began)and on Day 20 at end of study. Statistical significance was determinedusing unpaired nonparametric Mann-Whitney t-tests. Treatment withBSMUC16/CD3-001 was compared to the non-binding control (** p<0.01 for0.5 mg/kg, #p<0.05 for 0.1 mg/kg, $ p<0.05 for 0.01 mg/kgBSMUC16/CD3-001).

FIG. 7 shows the results of the OVCAR-3 model study 2 (Avg Radiance[p/s/cm²/sr] at Day 4). All groups had similar tumor burden as assessedby BLI before dosing started. Data shown is tumor burden as assessed byBLI on Day 4 post tumor implantation. Statistical significance wasdetermined using unpaired nonparametric Mann-Whitney t-tests. There wasno significant difference in tumor burden at Day 4 between groups.

FIG. 8 shows the results of the OVCAR-3 model study 2 (Avg Radiance[p/s/cm2²/sr] at Day 25). BSMUC16/CD3-005 significantly reduces tumorburden at 0.5, 1 and 5 mg/kg. NSG mice engrafted with human T cells wereimplanted with human OVCAR-3/Luc cells. Treatment began 5 days posttumor implantation. Mice were treated on Days 5, 8, 12, 15, 19, and 22with 0.1, 0.5, 1, or 5 mg/kg REGN4019 administered IV or administered aCD3-binding control or non-binding control (5 mg/kg IV). Data shown istumor burden as assessed by BLI on Day 25 post tumor implantation.Statistical significance was determined using unpaired nonparametricMann-Whitney t-tests. Treatment with BSMUC16/CD3-005 was compared to thenon-binding control (** p<0.01 for 5 mg/kg, ##p<0.01 for 1 mg/kg, $$p<0.01 for 0.5 mg/kg BSMUC16/CD3-005).

FIG. 9 shows the results of the OVCAR-3 model study 2 (Fold change inBLI-evident tumors between D4 and D25). BSMUC16/CD3-005 significantlyreduces tumor growth at 0.5, 1 and 5 mg/kg. NSG mice engrafted withhuman T cells were implanted with human OVCAR-3/Luc cells. Mice weretreated on Days 5, 8, 12, 15, 19, and 22 with 0.1, 0.5, 1, or 5 mg/kgREGN4019 administered IV or treated with a CD3-binding control ornon-binding control (5 mg/kg IV). Data shown is fold change in tumorburden from first measurement (taken the day before treatment began) andon Day 25, at end of study. Statistical significance was determinedusing unpaired nonparametric Mann-Whitney t-tests. Treatment withBSMUC16/CD3-005 was compared to the non-binding control (** p<0.01 for 5mg/kg, ##p<0.01 for 1 mg/kg, $$ p<0.01 for 0.5 mg/kg REGN4019).

FIG. 10 shows the results of the ID8-VEGF/huMUC16 model. Tumor size atDay 47 post implantation BSMUC16/CD3-001 significantly reduces tumorgrowth in a syngeneic model when treatment begins either on day ofimplantation or 10 days post tumor implantation. Mice expressing humanCD3 in place of mouse CD3 and a chimeric MUC16 molecule were implantedwith the murine ovarian tumor line expressing a portion of human MUC16.Mice were administered BSMUC16/CD3-001 (100 ug IP) on day ofimplantation or 10 days post implantation or administered CD3-bindingcontrol (100 ug IP) on day of implantation. Mice were treated on Days 0,4, 7, 10, 13, 17, 20 or 24 for immediate-treatment groups and on days10, 13, 17, 20 and 24 for the group where dosing started on D10. Datashown is tumor volume on Day 47 post implantation. Statisticalsignificance was determined using unpaired nonparametric Mann-Whitneyt-tests. Treatment with BSMUC16/CD3-001 was compared to the CD3-bindingcontrol (** p<0.01 starting at DO, * p<0.05 starting at D10).

FIGS. 11A-11C illustrate the results of flow cytometric analysis (orFACS) of the binding of select bispecific antibodies to PEO-1,OVCAR3-Luc, Jurkat cells, and cynomolgus T cells. The titration analysiswas done by testing a range of serial dilutions of each antibody: eitherMUC16×CD3 bispecific antibodies BSMUC16/CD3-001, BSMUC16/CD3-002, orBSMUC16/CD3-003, or a first or second isotype control antibody (havingno cross-reactivity to CD3 or MUC16).

FIGS. 12A and 12B depict examples of PEO-1 (FIG. 12A) or OVCAR3-Luc(FIG. 12B) cell killing in a 48 hour cytotoxicity assay followinganti-MUC16×anti-CD3 treatment in the presence of human PBMCs.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expression “CD3,” as used herein, refers to an antigen which isexpressed on T cells as part of the multimolecular T cell receptor (TCR)and which consists of a homodimer or heterodimer formed from theassociation of two of four receptor chains: CD3-epsilon, CD3-delta,CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acidsequence as set forth in SEQ ID NO:1897; human CD3-delta comprises theamino acid sequence as set forth in SEQ ID NO:1898. All references toproteins, polypeptides and protein fragments herein are intended torefer to the human version of the respective protein, polypeptide orprotein fragment unless explicitly specified as being from a non-humanspecies. Thus, the expression “CD3” means human CD3 unless specified asbeing from a non-human species, e.g., “mouse CD3,” “monkey CD3,” etc.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodiesand antigen-binding fragments of the present invention may bind solubleCD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

As used herein, the expression “cell surface-expressed CD3” means one ormore CD3 protein(s) that is/are expressed on the surface of a cell invitro or in vivo, such that at least a portion of a CD3 protein isexposed to the extracellular side of the cell membrane and is accessibleto an antigen-binding portion of an antibody. “Cell surface-expressedCD3” includes CD3 proteins contained within the context of a functionalT cell receptor in the membrane of a cell. The expression “cellsurface-expressed CD3” includes CD3 protein expressed as part of ahomodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon,delta/epsilon, and zeta/zeta CD3 dimers). The expression, “cellsurface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon,CD3-delta or CD3-gamma) that is expressed by itself, without other CD3chain types, on the surface of a cell. A “cell surface-expressed CD3”can comprise or consist of a CD3 protein expressed on the surface of acell which normally expresses CD3 protein. Alternatively, “cellsurface-expressed CD3” can comprise or consist of CD3 protein expressedon the surface of a cell that normally does not express human CD3 on itssurface but has been artificially engineered to express CD3 on itssurface.

The expression “MUC16,” as used herein, refers to mucin 16. MUC16 is asingle transmembrane domain highly glycosylated integral membraneglycoprotein that is highly expressed in ovarian cancer. The amino acidsequence of human MUC16 is set forth in SEQ ID NO:1899.

As used herein, “an antibody that binds MUC16” or an “anti-MUC16antibody” includes antibodies and antigen-binding fragments thereof thatspecifically recognize MUC16.

The term “antigen-binding molecule” includes antibodies andantigen-binding fragments of antibodies, including, e.g., bispecificantibodies.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., MUC16 or CD3). The term “antibody” includesimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (CL1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-MUC16 antibody oranti-CD3 antibody (or antigen-binding portion thereof) may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (Vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (XII) V_(L)-C_(H)1-C_(H)2-C_(H)3; (Xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-MUC16 monospecificantibodies or anti-MUC16/anti-CD3 bispecific antibodies of the inventionare human antibodies. The term “human antibody”, as used herein, isintended to include antibodies having variable and constant regionsderived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention also includes one-arm antibodies that bind MUC16.As used herein, a “one-arm antibody” means an antigen-binding moleculecomprising a single antibody heavy chain and a single antibody lightchain. The one-arm antibodies of the present invention may comprise anyof the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.

The anti-MUC16 or anti-MUC16/anti-CD3 antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes antibodies,and antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding (e.g., as measured by cell bindingtitration or FACS binding) or binding affinity (e.g., K_(D)), improvedor enhanced antagonistic or agonistic biological properties (as the casemay be), reduced immunogenicity, etc. Antibodies and antigen-bindingfragments obtained in this general manner are encompassed within thepresent invention.

The present invention also includes anti-MUC16 or anti-MUC16/anti-CD3antibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-MUC16 oranti-MUC16/anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acidsequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer,etc. conservative amino acid substitutions relative to any of the HCVR,LCVR, and/or CDR amino acid sequences set forth in Table 1 herein or asdescribed in Tables 16, 18, 19, 22, and 23 herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Germline Mutations

The anti-CD3 antibodies disclosed herein comprise one or more amino acidsubstitutions, insertions and/or deletions in the framework and/or CDRregions of the heavy chain variable domains as compared to thecorresponding germline sequences from which the antibodies were derived.

The present invention also includes antibodies, and antigen-bindingfragments thereof, which are derived from any of the amino acidsequences disclosed herein, wherein one or more amino acids within oneor more framework and/or CDR regions are mutated to the correspondingresidue(s) of the germline sequence from which the antibody was derived,or to the corresponding residue(s) of another human germline sequence,or to a conservative amino acid substitution of the correspondinggermline residue(s) (such sequence changes are referred to hereincollectively as “germline mutations”), and having weak or no detectablebinding to a CD3 antigen. Several such exemplary antibodies thatrecognize CD3 are described in Tables 16, 18, 19, 22, and 23 herein.

Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be tested for one or more desired properties such as, improvedbinding specificity, weak or reduced binding or binding affinity,improved or enhanced pharmacokinetic properties, reduced immunogenicity,etc. Antibodies and antigen-binding fragments obtained in this generalmanner given the guidance of the present disclosure are encompassedwithin the present invention.

The present invention also includes anti-CD3 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-CD3 antibodies having HCVR,LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 orfewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Tables 16, 18, 19, 22, and 23 herein. Theantibodies and bispecific antigen-binding molecules of the presentinvention comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived, while maintaining or improving the desired weak-to-nodetectable binding to CD3 antigen. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein, i.e. the amino acid substitutionmaintains or improves the desired weak to no detectable binding orbinding affinity in the case of anti-CD3 binding molecules. Examples ofgroups of amino acids that have side chains with similar chemicalproperties include (1) aliphatic side chains: glycine, alanine, valine,leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine andthreonine; (3) amide-containing side chains: asparagine and glutamine;(4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5)basic side chains: lysine, arginine, and histidine; (6) acidic sidechains: aspartate and glutamate, and (7) sulfur-containing side chainsare cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445. A “moderately conservative” replacement is any changehaving a nonnegative value in the PAM250 log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR and/or CDR amino acid sequencethat is substantially identical to any of the HCVR and/or CDR amino acidsequences disclosed herein, while maintaining or improving the desiredweak affinity to CD3 antigen. The term “substantial identity” or“substantially identical,” when referring to an amino acid sequencemeans that two amino acid sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least95% sequence identity, even more preferably at least 98% or 99% sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. In cases where two or moreamino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402.

Once obtained, antigen-binding domains that contain one or more germlinemutations were tested for decreased binding or binding affinityutilizing one or more in vitro assays. Although antibodies thatrecognize a particular antigen are typically screened for their purposeby testing for high (i.e. strong) binding or binding affinity to theantigen, the antibodies of the present invention exhibit weak binding orno detectable binding. Bispecific antigen-binding molecules comprisingone or more antigen-binding domains obtained in this general manner arealso encompassed within the present invention and were found to beadvantageous as avidity-driven tumor therapies.

Unexpected benefits, for example, improved pharmacokinetic propertiesand low toxicity to the patient may be realized from the methodsdescribed herein.

Binding Properties of the Antibodies

As used herein, the term “binding” in the context of the binding of anantibody, immunoglobulin, antibody-binding fragment, or Fc-containingprotein to either, e.g., a predetermined antigen, such as a cell surfaceprotein or fragment thereof, typically refers to an interaction orassociation between a minimum of two entities or molecular structures,such as an antibody-antigen interaction.

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore 3000 instrument using the antigen as theligand and the antibody, Ig, antibody-binding fragment, or Fc-containingprotein as the analyte (or antiligand). Cell-based binding strategies,such as fluorescent-activated cell sorting (FACS) binding assays, arealso routinely used, and FACS data correlates well with other methodssuch as radioligand competition binding and SPR (Benedict, Calif., JImmunol Methods. 1997, 201(2):223-31; Geuijen, C A, et al. J ImmunolMethods. 2005, 302(1-2):68-77).

Accordingly, the antibody or antigen-binding protein of the inventionbinds to the predetermined antigen or cell surface molecule (receptor)having an affinity corresponding to a K_(D) value that is at leastten-fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein). According to the present invention, the affinity ofan antibody corresponding to a K_(D) value that is equal to or less thanten-fold lower than a non-specific antigen may be considerednon-detectable binding, however such an antibody may be paired with asecond antigen binding arm for the production of a bispecific antibodyof the invention.

The term “K_(D)” (M) refers to the dissociation equilibrium constant ofa particular antibody-antigen interaction, or the dissociationequilibrium constant of an antibody or antibody-binding fragment bindingto an antigen. There is an inverse relationship between K_(D) andbinding affinity, therefore the smaller the K_(D) value, the higher,i.e. stronger, the affinity. Thus, the terms “higher affinity” or“stronger affinity” relate to a higher ability to form an interactionand therefore a smaller K_(D) value, and conversely the terms “loweraffinity” or “weaker affinity” relate to a lower ability to form aninteraction and therefore a larger K_(D) value. In some circumstances, ahigher binding affinity (or K_(D)) of a particular molecule (e.g.antibody) to its interactive partner molecule (e.g. antigen X) comparedto the binding affinity of the molecule (e.g. antibody) to anotherinteractive partner molecule (e.g. antigen Y) may be expressed as abinding ratio determined by dividing the larger K_(D) value (lower, orweaker, affinity) by the smaller K_(D) (higher, or stronger, affinity),for example expressed as 5-fold or 10-fold greater binding affinity, asthe case may be.

The term “k_(d)” (sec-1 or 1/s) refers to the dissociation rate constantof a particular antibody-antigen interaction, or the dissociation rateconstant of an antibody or antibody-binding fragment. Said value is alsoreferred to as the k_(off) value.

The term “k_(a)” (M-1×sec-1 or 1/M) refers to the association rateconstant of a particular antibody-antigen interaction, or theassociation rate constant of an antibody or antibody-binding fragment.

The term “K_(A)” (M-1 or 1/M) refers to the association equilibriumconstant of a particular antibody-antigen interaction, or theassociation equilibrium constant of an antibody or antibody-bindingfragment. The association equilibrium constant is obtained by dividingthe k_(a) by the k_(d).

The term “EC50” or “EC₅₀” refers to the half maximal effectiveconcentration, which includes the concentration of an antibody whichinduces a response halfway between the baseline and maximum after aspecified exposure time. The EC₅₀ essentially represents theconcentration of an antibody where 50% of its maximal effect isobserved. In certain embodiments, the EC₅₀ value equals theconcentration of an antibody of the invention that gives half-maximalbinding to cells expressing CD3 or tumor-associated antigen, asdetermined by e.g. a FACS binding assay. Thus, reduced or weaker bindingis observed with an increased EC₅₀, or half maximal effectiveconcentration value.

In one embodiment, decreased binding can be defined as an increased EC₅₀antibody concentration which enables binding to the half-maximal amountof target cells.

In another embodiment, the EC₅₀ value represents the concentration of anantibody of the invention that elicits half-maximal depletion of targetcells by T cell cytotoxic activity. Thus, increased cytotoxic activity(e.g. T cell-mediated tumor cell killing) is observed with a decreasedEC₅₀, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-MUC16 monospecific antibodies oranti-MUC16/anti-CD3 bispecific antibodies of the present invention canbe linked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second or additionalbinding specificity.

Use of the expression “anti-CD3 antibody” or “anti-MUC16 antibody”herein is intended to include both monospecific anti-CD3 or anti-MUC16antibodies as well as bispecific antibodies comprising a CD3-binding armand a MUC16-binding arm. Thus, the present invention includes bispecificantibodies wherein one arm of an immunoglobulin binds human CD3, and theother arm of the immunoglobulin is specific for human MUC16. TheCD3-binding arm can comprise any of the HCVR/LCVR or CDR amino acidsequences as set forth in Tables 1, 16, 18, 19, 22, and 23 herein.

In certain embodiments, the CD3-binding arm binds to human CD3 andinduces human T cell activation. In certain embodiments, the CD3-bindingarm binds weakly to human CD3 and induces human T cell activation. Inother embodiments, the CD3-binding arm binds weakly to human CD3 andinduces tumor-associated antigen-expressing cell killing in the contextof a bispecific or multispecific antibody. In other embodiments, theCD3-binding arm binds or associated weakly with human and cynomolgus(monkey) CD3, yet the binding interaction is not detectable by in vitroassays known in the art. The MUC16-binding arm can comprise any of theHCVR/LCVR or CDR amino acid sequences as set forth in Table 1 herein.

According to certain exemplary embodiments, the present inventionincludes bispecific antigen-binding molecules that specifically bind CD3and MUC16. Such molecules may be referred to herein as, e.g.,“anti-CD3/anti-MUC16,” or “anti-CD3×MUC16” or “CD3×MUC16” bispecificmolecules, or other similar terminology (e.g., anti-MUC16/anti-CD3). Theinvention provides bispecific antigen-binding molecules constructed witha first antigen-binding arm that binds MUC16 and a secondantigen-binding arm that binds CD3. In some embodiments, the anti-CD3arm comprises a heavy chain derived from IGHV3-9*01, IGHJ6*02,IGHD5-12*01. In other embodiments, the bispecific antigen-bindingmolecule activates human PBMC cells and/or induces cytotoxic activity ontumor antigen-expressing cell lines.

The term “MUC16,” as used herein, refers to the human MUC16 proteinunless specified as being from a non-human species (e.g., “mouse MUC16,”“monkey MUC16,” etc.). The human MUC16 protein has the amino acidsequence shown in SEQ ID NO:1899.

The aforementioned bispecific antigen-binding molecules thatspecifically bind CD3 and MUC16 may comprise an anti-CD3 antigen-bindingmolecule which binds to CD3 with a weak binding affinity such asexhibiting a K_(D) of greater than about 40 nM, as measured by an invitro affinity binding assay. The aforementioned bispecificantigen-binding molecules may comprise an anti-CD3 antigen-bindingmolecule which binds to CD3 and exhibits an EC50 of greater than about100 nM, as measured by a FACS titration assay. The aforementionedbispecific antigen-binding molecules may comprise an anti-CD3antigen-binding molecule which exhibits no measurable or observablebinding to CD3, as measured by an in vitro affinity binding assay or aFACS titration assay, yet retains ability to activate human PBMC cellsand/or induce cytotoxic activity on tumor antigen-expressing cell lines.

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., CD3),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., MUC16).

In certain exemplary embodiments of the present invention, thebispecific antigen-binding molecule is a bispecific antibody. Eachantigen-binding domain of a bispecific antibody comprises a heavy chainvariable domain (HCVR) and a light chain variable domain (LCVR). In thecontext of a bispecific antigen-binding molecule comprising a first anda second antigen-binding domain (e.g., a bispecific antibody), the CDRsof the first antigen-binding domain may be designated with the prefix“A1” and the CDRs of the second antigen-binding domain may be designatedwith the prefix “A2”. Thus, the CDRs of the first antigen-binding domainmay be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and theCDRs of the second antigen-binding domain may be referred to herein asA2-HCDR1, A2-HCDR2, and A2-HCDR3.

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abispecific antigen-binding molecule of the present invention.Alternatively, the first antigen-binding domain and the secondantigen-binding domain may each be connected to a separate multimerizingdomain. The association of one multimerizing domain with anothermultimerizing domain facilitates the association between the twoantigen-binding domains, thereby forming a bispecific antigen-bindingmolecule. As used herein, a “multimerizing domain” is any macromolecule,protein, polypeptide, peptide, or amino acid that has the ability toassociate with a second multimerizing domain of the same or similarstructure or constitution. For example, a multimerizing domain may be apolypeptide comprising an immunoglobulin C_(H)3 domain. A non-limitingexample of a multimerizing component is an Fc portion of animmunoglobulin (comprising a C_(H)2-C_(H)3 domain), e.g., an Fc domainof an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as wellas any allotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of from 1 to about 200 amino acids in lengthcontaining at least one cysteine residue. In other embodiments, themultimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bispecificantigen-binding molecule. Specific exemplary bispecific formats that canbe used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody,IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab² bispecific formats (see,e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,for a review of the foregoing formats).

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q orK) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g.,434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes bispecific antigen-binding moleculescomprising a first C_(H)3 domain and a second Ig C_(H)3 domain, whereinthe first and second Ig C_(H)3 domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No.8,586,713. Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies.

In certain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype. Forexample, a chimeric Fc domain can comprise part or all of a C_(H)2sequence derived from a human IgG1, human IgG2 or human IgG4 C_(H)2region, and part or all of a C_(H)3 sequence derived from a human IgG1,human IgG2 or human IgG4. A chimeric Fc domain can also contain achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” sequence, derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence, derivedfrom a human IgG1, a human IgG2 or a human IgG4 hinge region. Aparticular example of a chimeric Fc domain that can be included in anyof the antigen-binding molecules set forth herein comprises, from N- toC-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4C_(H)2]-[IgG4 C_(H)3]. Another example of a chimeric Fc domain that canbe included in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upper hinge]-[IgG2lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimericFc domains that can be included in any of the antigen-binding moleculesof the present invention are described in US Publication 2014/0243504,published Aug. 28, 2014, which is herein incorporated in its entirety.Chimeric Fc domains having these general structural arrangements, andvariants thereof, can have altered Fc receptor binding, which in turnaffects Fc effector function.

In certain embodiments, the invention provides an antibody heavy chainwherein the heavy chain constant region (CH) region comprises an aminoacid sequence at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identical to any one of SEQ ID NO: 1911, SEQ ID NO: 1912, SEQID NO: 1913, SEQ ID NO: 1914, SEQ ID NO: 1915, SEQ ID NO: 1916, SEQ IDNO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919 or SEQ ID NO: 1920. In someembodiments, the heavy chain constant region (CH) region comprises anamino acid sequence selected from the group consisting of SEQ ID NO:1911, SEQ ID NO: 1912, SEQ ID NO: 1913, SEQ ID NO: 1914, SEQ ID NO:1915, SEQ ID NO: 1916, SEQ ID NO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919and SEQ ID NO: 1920.

In other embodiments, the invention provides an antibody heavy chainwherein the Fc domain comprises an amino acid sequence at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identical to any oneof SEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923 SEQ ID NO: 1924 SEQID NO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928, SEQ IDNO: 1929 or SEQ ID NO: 1930. In some embodiments, the Fc domaincomprises an amino acid sequence selected form the group consisting ofSEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923 SEQ ID NO: 1924 SEQ IDNO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928, SEQ ID NO:1929 and SEQ ID NO: 1930.

Sequence Variants

The antibodies and bispecific antigen-binding molecules of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. Theantigen-binding molecules of the present invention may compriseantigen-binding domains which are derived from any of the exemplaryamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antigen-binding domain wasoriginally derived. In other embodiments, only certain residues aremutated back to the original germline sequence, e.g., only the mutatedresidues found within the first 8 amino acids of FR1 or within the last8 amino acids of FR4, or only the mutated residues found within CDR1,CDR2 or CDR3. In other embodiments, one or more of the framework and/orCDR residue(s) are mutated to the corresponding residue(s) of adifferent germline sequence (i.e., a germline sequence that is differentfrom the germline sequence from which the antigen-binding domain wasoriginally derived). Furthermore, the antigen-binding domains maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germlinesequence while certain other residues that differ from the originalgermline sequence are maintained or are mutated to the correspondingresidue of a different germline sequence. Once obtained, antigen-bindingdomains that contain one or more germline mutations can be easily testedfor one or more desired property such as, improved binding specificity,increased binding or binding affinity, improved or enhanced antagonisticor agonistic biological properties (as the case may be), reducedimmunogenicity, etc. Bispecific antigen-binding molecules comprising oneor more antigen-binding domains obtained in this general manner areencompassed within the present invention.

The present invention also includes antigen-binding molecules whereinone or both antigen-binding domains comprise variants of any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein having oneor more conservative substitutions. For example, the present inventionincludes antigen-binding molecules comprising an antigen-binding domainhaving HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 orfewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. A “conservative amino acid substitution” isone in which an amino acid residue is substituted by another amino acidresidue having a side chain (R group) with similar chemical properties(e.g., charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. Examples of groups of amino acids that have side chains withsimilar chemical properties include (1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl sidechains: serine and threonine; (3) amide-containing side chains:asparagine and glutamine; (4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, andhistidine; (6) acidic side chains: aspartate and glutamate, and (7)sulfur-containing side chains are cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443-1445, herein incorporated by reference.A “moderately conservative” replacement is any change having anonnegative value in the PAM250 log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR, LCVR, and/or CDR amino acidsequence that is substantially identical to any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein. The term “substantialidentity” or “substantially identical,” when referring to an amino acidsequence means that two amino acid sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 95% sequence identity, even more preferably at least 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. In cases where two ormore amino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, herein incorporated by reference.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-MUC16 antibodies, andanti-CD3/anti-MUC16 bispecific antigen-binding molecules, withpH-dependent binding characteristics. For example, an anti-MUC16antibody of the present invention may exhibit reduced binding to MUC16at acidic pH as compared to neutral pH. Alternatively, anti-MUC16antibodies of the invention may exhibit enhanced binding to MUC16 atacidic pH as compared to neutral pH. The expression “acidic pH” includespH values less than about 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8,5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15,5.1, 5.05, 5.0, or less. As used herein, the expression “neutral pH”means a pH of about 7.0 to about 7.4. The expression “neutral pH”includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35,and 7.4.

In certain instances, “reduced binding . . . at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of theantibody binding to its antigen at acidic pH to the K_(D) value of theantibody binding to its antigen at neutral pH (or vice versa). Forexample, an antibody or antigen-binding fragment thereof may be regardedas exhibiting “reduced binding to MUC16 at acidic pH as compared toneutral pH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-MUC16antibodies, and anti-CD3/anti-MUC16 bispecific antigen-bindingmolecules, are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes antibodies comprising a mutation in theC_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the present invention includes anti-MUC16 antibodies, andanti-CD3/anti-MUC16 bispecific antigen-binding molecules, comprising anFc domain comprising one or more pairs or groups of mutations selectedfrom the group consisting of: 250Q and 248L (e.g., T250Q and M248L);252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g.,M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Allpossible combinations of the foregoing Fc domain mutations, and othermutations within the antibody variable domains disclosed herein, arecontemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and BispecificAntigen-Binding Molecules

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human MUC16 with high affinity (e.g., sub-nanomolarK_(D) values).

According to certain embodiments, the present invention includesantibodies and antigen-binding fragments of antibodies that bind humanMUC16 (e.g., at 25° C.) with a K_(D) of less than about 60 nM asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Example 4 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind MUC16 with aK_(D) of less than about 60 nM, less than about 40 nM, less than about20 nM, less than about 10 nM, less than about 8 nM, less than about 7nM, less than about 6 nM, less than about 5 nM, less than about 4 nM,less than about 3 nM, less than about 2 nM, less than about 1 nM, lessthan about 800 pM, less than about 700 pM, less than about 500 pM, lessthan about 400 pM, or less than about 300 pM, as measured by surfaceplasmon resonance, e.g., using an assay format as defined in Example 4herein (e.g., mAb-capture or antigen-capture format), or a substantiallysimilar assay. The present invention includes bispecific antigen-bindingmolecules (e.g., bispecific antibodies which bind MUC16 with a K_(D) ofless than about 7 nM, as measured by surface plasmon resonance, e.g.,using an assay format as defined in Example 4 herein (e.g., mAb-captureor antigen-capture format), or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind MUC16 with a dissociative half-life (t½) ofgreater than about 10 minutes or greater than about 125 minutes asmeasured by surface plasmon resonance at 25° C., e.g., using an assayformat as defined in Example 4 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind MUC16 with a t½ of greater than about 10minutes, greater than about 20 minutes, greater than about 30 minutes,greater than about 40 minutes, greater than about 50 minutes, greaterthan about 60 minutes, greater than about 70 minutes, greater than about80 minutes, greater than about 90 minutes, greater than about 100minutes, greater than about 110 minutes, or greater than about 120minutes, as measured by surface plasmon resonance at 25° C., e.g., usingan assay format as defined in Example 4 herein (e.g., mAb-capture orantigen-capture format), or a substantially similar assay. The presentinvention includes bispecific antigen-binding molecules (e.g.,bispecific antibodies which bind MUC16 with a of greater than about 10minutes or greater than about 20 minutes as measured by surface plasmonresonance at 25° C., e.g., using an assay format as defined in Example 4herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof which bind specifically to human cell lines whichexpress endogenous MUC16 (e.g., OVCAR-3), as determined by anelectrochemoluminescence-based detection assay as set forth in Example 2or a substantially similar assay.

The present invention also includes anti-CD3/anti-MUC16 bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of: (a) inhibiting tumor growth inimmunocompromised mice bearing human ovarian cancer xenografts; and (b);suppressing tumor growth of established tumors in immunocompromised micebearing human ovarian cancer xenografts (see, e.g., Example 8).

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with high affinity. The present inventionalso includes antibodies and antigen-binding fragments thereof that bindhuman CD3 with medium or low affinity, depending on the therapeuticcontext and particular targeting properties that are desired. In somecases, the low affinity includes antibodies that bind CD3 with a K_(D)or EC₅₀ (e.g., as measured in a surface plasmon resonance assay) ofgreater than 300 nM, greater than 500 nM or greater than 1 μM. Thepresent invention also includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with no measureable affinity. For example,in the context of a bispecific antigen-binding molecule, wherein one armbinds CD3 and another arm binds a target antigen (e.g., MUC16), it maybe desirable for the target antigen-binding arm to bind the targetantigen with high affinity while the anti-CD3 arm binds CD3 with onlymoderate or low affinity or no affinity. In this manner, preferentialtargeting of the antigen-binding molecule to cells expressing the targetantigen may be achieved while avoiding general/untargeted CD3 bindingand the consequent adverse side effects associated therewith.

The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies) which are capable of simultaneouslybinding to human CD3 and a human MUC16. The binding arm that interactswith cells that express CD3 may have weak to no detectable binding asmeasured in a suitable in vitro binding assay. The extent to which abispecific antigen-binding molecule binds cells that express CD3 and/orMUC16 can be assessed by fluorescence activated cell sorting (FACS), asillustrated in Example 5 herein.

For example, the present invention includes antibodies, antigen-bindingfragments, and bispecific antibodies thereof which specifically bindhuman T-cell lines which express CD3 but do not express MUC16 (e.g.,Jurkat), primate T-cells (e.g., cynomolgus peripheral blood mononuclearcells [PBMCs]), and/or MUC16-expressing cells.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 with weak (i.e.low) or even no detectable binding or binding affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind monkey (i.e. cynomolgus) CD3with weak (i.e. low) or even no detectable binding or binding affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 and induce T cellactivation.

The present invention includes anti-CD3/anti-MUC16 bispecificantigen-binding molecules which are capable of depleting tumorantigen-expressing cells in a subject (see, e.g., Example 8, in abioluminescent imaging assay, or a substantially similar assay). Forexample, according to certain embodiments, anti-CD3/anti-MUC16bispecific antigen-binding molecules are provided, wherein a singleadministration of 10 μg of the bispecific antigen-binding molecule to asubject causes a reduction in the number of MUC16-expressing cells inthe subject (e.g., tumor growth in the subject is suppressed orinhibited). Unless otherwise indicated, bioluminescent radiance refersto [p/s/cm2²/sr].

The present invention also includes anti-MUC16 antibody drug conjugateswhich inhibit tumor growth in in vivo MUC16 positive ovarian cancerxenograft models (see, e.g., Example 10, in a bioluminescent imagingassay, or a substantially similar assay). In certain embodiments,anti-MUC16 antibody drug conjugates with Compound 7 are provided whereinfour once weekly doses administered at a dose of 85 μg/kg inhibitintraperitoneal OVCAR3/luc tumor growth in in vivo. In certainembodiments, anti-MUC16 antibody drug conjugates with Compound 7 areprovided wherein four once weekly doses administered at a dose of 85μg/kg inhibit subcutaneous OVCAR3/luc tumor growth in in vivo. Incertain embodiments, anti-MUC16 antibody drug conjugates with Compound10 are provided wherein a single dose at a dose of 85 μg/kg, 170 μg/kg,or 340 μg/kg inhibit intraperitoneal OVCAR3/luc tumor growth in in vivo.Unless otherwise indicated, bioluminescent radiance refers to[p/s/cm2²/sr].

The present invention also includes anti-CD3/anti-MUC16 bispecificantigen-binding molecules which exhibit pharmacokinetic profiles inhumanized MUC16×CD3 mice (mice homozygous for human MUC16 and CD3expression, MUC16^(hu/hu)×CD3 hu/hu) CD3 humanized mice (mice homozygousfor human CD3 expression, CD3^(hu/hu)) and strain-matched (75% C57BL,25%129Sv) wild-type (WT) mice, as described in Example 7 and shown inFIGS. 1, 2, and 3 .

Epitope Mapping and Related Technologies

The epitope on CD3 and/or MUC16 to which the antigen-binding moleculesof the present invention bind may consist of a single contiguoussequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) amino acids of a CD3 or MUC16 protein.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) of CD3 or MUC16. The antibodies ofthe invention may interact with amino acids contained within a singleCD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or may interactwith amino acids on two or more different CD3 chains. The term“epitope,” as used herein, refers to an antigenic determinant thatinteracts with a specific antigen binding site in the variable region ofan antibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstances, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of an antibody“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques include, e.g., routine cross-blockingassay such as that described Antibodies, Harlow and Lane (Cold SpringHarbor Press, Cold Spring Harb., N.Y.), alanine scanning mutationalanalysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol248:443-463), and peptide cleavage analysis. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer, 2000, Protein Science 9:487-496).Another method that can be used to identify the amino acids within apolypeptide with which an antigen-binding domain of an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

The present invention further includes anti-MUC16 antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. antibodies comprising any of the amino acidsequences as set forth in Table 1 herein). Likewise, the presentinvention also includes anti-MUC16 antibodies that compete for bindingto MUC16 with any of the specific exemplary antibodies described herein(e.g. antibodies comprising any of the amino acid sequences as set forthin Table 1 herein).

The present invention also includes bispecific antigen-binding moleculescomprising a first antigen-binding domain that specifically binds humanCD3 and/or cynomolgus CD3 with low or no detectable binding or bindingaffinity, and a second antigen binding domain that specifically bindshuman MUC16, wherein the first antigen-binding domain binds to the sameepitope on CD3 as any of the specific exemplary CD3-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain binds to the same epitope on MUC16 as any of thespecific exemplary MUC16-specific antigen-binding domains describedherein.

Likewise, the present invention also includes bispecific antigen-bindingmolecules comprising a first antigen-binding domain that specificallybinds human CD3, and a second antigen binding domain that specificallybinds human MUC16, wherein the first antigen-binding domain competes forbinding to CD3 with any of the specific exemplary CD3-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain competes for binding to MUC16 with any of thespecific exemplary MUC16-specific antigen-binding domains describedherein.

One can easily determine whether a particular antigen-binding molecule(e.g., antibody) or antigen-binding domain thereof binds to the sameepitope as, or competes for binding with, a reference antigen-bindingmolecule of the present invention by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope on MUC16 (or CD3) as a reference bispecific antigen-bindingmolecule of the present invention, the reference bispecific molecule isfirst allowed to bind to a MUC16 protein (or CD3 protein). Next, theability of a test antibody to bind to the MUC16 (or CD3) molecule isassessed. If the test antibody is able to bind to MUC16 (or CD3)following saturation binding with the reference bispecificantigen-binding molecule, it can be concluded that the test antibodybinds to a different epitope of MUC16 (or CD3) than the referencebispecific antigen-binding molecule. On the other hand, if the testantibody is not able to bind to the MUC16 (or CD3) molecule followingsaturation binding with the reference bispecific antigen-bindingmolecule, then the test antibody may bind to the same epitope of MUC16(or CD3) as the epitope bound by the reference bispecificantigen-binding molecule of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference bispecific antigen-binding molecule or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antigen-binding proteins bind to the same (oroverlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess ofone antigen-binding protein inhibits binding of the other by at least50% but preferably 75%, 90% or even 99% as measured in a competitivebinding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antigen-binding proteins aredeemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantigen-binding protein reduce or eliminate binding of the other. Twoantigen-binding proteins are deemed to have “overlapping epitopes” ifonly a subset of the amino acid mutations that reduce or eliminatebinding of one antigen-binding protein reduce or eliminate binding ofthe other.

To determine if an antibody or antigen-binding domain thereof competesfor binding with a reference antigen-binding molecule, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding molecule is allowedto bind to a MUC16 protein (or CD3 protein) under saturating conditionsfollowed by assessment of binding of the test antibody to the MUC16 (orCD3) molecule. In a second orientation, the test antibody is allowed tobind to a MUC16 (or CD3) molecule under saturating conditions followedby assessment of binding of the reference antigen-binding molecule tothe MUC16 (or CD3) molecule. If, in both orientations, only the first(saturating) antigen-binding molecule is capable of binding to the MUC16(or CD3) molecule, then it is concluded that the test antibody and thereference antigen-binding molecule compete for binding to MUC16 (orCD3). As will be appreciated by a person of ordinary skill in the art,an antibody that competes for binding with a reference antigen-bindingmolecule may not necessarily bind to the same epitope as the referenceantibody, but may sterically block binding of the reference antibody bybinding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,two different antigen-binding domains, specific for two differentantigens (e.g., CD3 and MUC16), can be appropriately arranged relativeto one another to produce a bispecific antigen-binding molecule of thepresent invention using routine methods. (A discussion of exemplarybispecific antibody formats that can be used to construct the bispecificantigen-binding molecules of the present invention is provided elsewhereherein). In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains) of the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making such antibodiesare well known in the art. For example, one or more of the heavy and/orlight chains of the bispecific antigen-binding molecules of the presentinvention can be prepared using VELOCIMMUNE™ technology. UsingVELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD3 or MUC16) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

Genetically engineered animals may be used to make human bispecificantigen-binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human bispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454). Fully human refers to anantibody, or antigen-binding fragment or immunoglobulin domain thereof,comprising an amino acid sequence encoded by a DNA derived from a humansequence over the entire length of each polypeptide of the antibody orantigen-binding fragment or immunoglobulin domain thereof. In someinstances, the fully human sequence is derived from a protein endogenousto a human. In other instances, the fully human protein or proteinsequence comprises a chimeric sequence wherein each component sequenceis derived from human sequence. While not being bound by any one theory,chimeric proteins or chimeric sequences are generally designed tominimize the creation of immunogenic epitopes in the junctions ofcomponent sequences, e.g. compared to any wild-type human immunoglobulinregions or domains.

Bioequivalents

The present invention encompasses antigen-binding molecules having aminoacid sequences that vary from those of the exemplary molecules disclosedherein but that retain the ability to bind CD3 and/or MUC16. Suchvariant molecules may comprise one or more additions, deletions, orsubstitutions of amino acids when compared to parent sequence, butexhibit biological activity that is essentially equivalent to that ofthe described bispecific antigen-binding molecules.

The present invention includes antigen-binding molecules that arebioequivalent to any of the exemplary antigen-binding molecules setforth herein. Two antigen-binding proteins, or antibodies, areconsidered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledoes or multiple dose. Some antigen-binding proteins will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antigen-binding protein.

Bioequivalent variants of the exemplary bispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antigen-binding proteinsmay include variants of the exemplary bispecific antigen-bindingmolecules set forth herein comprising amino acid changes which modifythe glycosylation characteristics of the molecules, e.g., mutationswhich eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, antigen-bindingmolecules are provided which bind to human CD3 but not to CD3 from otherspecies. Also provided are antigen-binding molecules which bind to humanMUC16. The present invention also includes antigen-binding moleculesthat bind to human CD3 and to CD3 from one or more non-human species;and/or antigen-binding molecules that bind to human MUC16.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provided which bind to human CD3 and/orhuman MUC16 and may bind or not bind, as the case may be, to one or moreof mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CD3and/or MUC16. For example, in a particular exemplary embodiment of thepresent invention bispecific antigen-binding molecules are providedcomprising a first antigen-binding domain that binds human CD3 andcynomolgus CD3, and a second antigen-binding domain that specificallybinds human MUC16.

Antibody-Drug Conjugates (ADCs)

The present invention provides antibody-drug conjugates (ADCs)comprising an anti-MUC16 antibody or antigen-binding fragment thereofconjugated to a therapeutic moiety such as a cytotoxic agent, achemotherapeutic drug, immunosuppressant or a radioisotope. In generalterms, the ADCs comprise: A-[L-P]_(y), in which A is an antigen-bindingmolecule, e.g. an anti-MUC16 antibody, or a fragment thereof (e.g., afragment comprising at least a HCDR3 selected from any of the HCDR3amino acid sequences listed in Table 1), L is a linker, P is the payloador therapeutic moiety (e.g., cytotoxic agent), and y is an integer from1 to 30. In various embodiments, the ADC comprises an anti-MUC16antibody or antigen-binding fragment thereof that comprises the CDRs ofa HCVR and a LCVR having the amino acid sequences of the SEQ ID NOs(e.g., SEQ ID NOs: 2 and 10) set forth in Table 1, or specific HCVR/LCVRpairs (e.g., SEQ ID NOs: 2/10). In some cases, the anti-MUC16 antibodyor fragment comprises CDRs with the amino acid sequences of the SEQ IDNOs (e.g., SEQ ID NOs: 4-6-8-12-14-16) set forth in Table 1. In somecases, the anti-MUC16 antibody or fragment comprises a HCVR and a LCVRhaving the amino acid sequences of the SEQ ID NOs (e.g., SEQ ID NOs: 2and 10) set forth in Table 1, or specific amino acid sequence pairs(e.g., SEQ ID NOs: 2/10). In some cases, the anti-MUC16 antibody is anantibody or antigen-binding fragment that binds human MUC16 within oneor more of five membrane-proximal SEA domains of human MUC16corresponding to residues 13791-14451 of SEQ ID NO: 1899. In some cases,the anti-MUC16 antibody is an antibody or antigen-binding fragment thatbinds human MUC16 within residues 13810-14451 of SEQ ID NO: 1899. Insome cases, the anti-MUC16 antibody is an antibody or antigen-bindingfragment that binds to any one of more of SEA1, SEA2, SEA3, SEA4, SEA5,SEA6, SEA7, SEAR, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15 orSEA16 of human MUC16.

Cytotoxic agents include any agent that is detrimental to the growth,viability or propagation of cells. The antigen-binding molecules orantibodies of the invention deliver these cytotoxic agents, referred toherein as “payloads”, to the target cells. Examples of suitablecytotoxic agents and chemotherapeutic agents for forming ADCs are knownin the art.

Examples of suitable cytotoxic agents and chemotherapeutic agents thatcan be conjugated to anti-MUC16 antibodies in accordance with thisaspect of the invention include, e.g.,1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins (monomethyl auristatin Eor monomethyl auristatin F), bleomycin, busulfan, butyric acid,calicheamicins, camptothecin, carminomycins, carmustine, cemadotins,cisplatin, colchicin, combretastatins, cyclophosphamide, cytarabine,cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins,eleutherobins, emetine, epothilones, esperamicin, estramustines,ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D,glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, rhizoxins, streptozotocin,tallysomycins, taxol, tenoposide, tetracaine, thioepa chlorambucil,tomaymycins, topotecans, tubulysin, vinblastine, vincristine, vindesine,vinorelbines, and derivatives of any of the foregoing.

According to certain embodiments, the cytotoxic agent that is conjugatedto an anti-MUC16 antibody is an auristatin such as monomethyl auristatinE (MMAE) or monomethyl auristatin F (MMAF), a tubulysin such as TUB-OHor TUB-OMOM, a tomaymycin derivative, a dolastatin derivative, or amaytansinoid such as DM1 or DM4. In some embodiments, the cytotoxicagent is a maytansinoid having the structure of Formula (I), includingstereoisomers of the compounds of Formula (I):

wherein A is arylene or heteroarylene.

In some embodiments, A is a divalent radical of benzene, of pyridine, ofnaphthalene, or of quinolone, which are optionally substituted.

In some embodiments, A is arylene.

In some embodiments, A is:

wherein:

-   -   R¹ is, independently at each occurrence, alkyl, alkenyl,        alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl,        heterocycloalkyl, hydroxyl, cyano, nitro,

-   -    or azido,        -   wherein R^(A) is alkyl or heteroalkyl;    -   n is an integer from 0 to 4;    -   m is and integer from 0 to 3;    -   p is an integer from 0 to 6; and    -   q is an integer from 0 to 5.

In some embodiments, the compound of Formula I is selected from thegroup consisting of:

In one embodiment, the compound of Formula (I) is:

In some embodiments, the maytansinoid of Formula (I) is conjugated to ananti-MUC16 antibody or antigen-binding fragment thereof via a linker, asshown in Formula (IA), below:

wherein:

-   -   A is arylene or heteroarylene, as discussed above in connection        with Formula (I);    -   L is a linker;    -   BA is an anti-MUC16 antibody or antigen-binding fragment        thereof; and    -   k is an integer from 1 to 30.

In various embodiments, L is:

wherein:

-   -   SP is a spacer;

is one or more bonds to the anti-MUC16 antibody or fragment thereof;

-   -   AA¹ is an amino acid; and    -   AA² is an amino acid.

In some embodiments, AA¹-AA² is: valine-citrulline, citrulline-valine,lysine-phenylalanine, phenylalanine-lysine, valine-asparagine,asparagine-valine, threonine-asparagine, asparagine-threonine,serine-asparagine, asparagine-serine, phenylalanine-asparagine,asparagine-phenylalanine, leucine-asparagine, asparagine-leucine,isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine,asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid,citrulline-asparagine, asparagine-citrulline, alanine-asparagine, orasparagine-alanine.

In some embodiments, SP is:

wherein:

is a bond to the anti-MUC16 antibody or fragment thereof; and

-   -   b is an integer from 2 to 8.        In other embodiments, L is:

wherein:

is a bond to the anti-MUC16 antibody or fragment thereof; and

-   -   b is an integer from 2 to 8.

In one embodiment, the compound of Formula (IA), including the linker,that is bound to the anti-MUC16 antibody or antigen-binding fragmentthereof is:

wherein

is a bond to the anti-MUC16 antibody or fragment thereof. In someinstances, this moiety is referred to as “Compound 10.”

In one embodiment, the compound of Formula (IA), including the linker,that is bound to the anti-MUC16 antibody or antigen-binding fragmentthereof is:

wherein

is a bond to the anti-MUC16 antibody or fragment thereof. In someinstances, this moiety is referred to as “Compound 60.”

In some embodiments, the cytotoxic agent is a maytansinoid having thestructure of Formula (II), including stereoisomers of the compounds ofFormula (II):

wherein:

-   -   A_(3a) is an amino acid, a peptide having 2-20 amino acids, an        alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a        heteroaryl, a heterocyclyl, —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—,        —C(═O)—O—, —O—C(═O)—O—, —C(═O)—(CH_(x))_(p1)—,        —C(═O)—O—(CH_(x))_(p1)—, —(CH_(x))_(p1)—C(═O)—,        —(CH_(x))_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,        —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—,        —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,        —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,        —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein        alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and        heterocyclyl are optionally substituted; and        -   p1, p2 and p3 are each independently 0, or an integer from 1            to 100;        -   x is 0, 1 or 2;        -   R₄, R₅, R₆ and R₈ are each independently H, or a substituted            or unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl,            or heterocyclyl; and        -   R_(4a) is a substituted or unsubstituted: alkyl, alkenyl,            alkynyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, the compound of Formula (II) is selected from thegroup consisting of:

In one embodiment, the compound of Formula (II) is:

In some embodiments, the maytansinoid of Formula (II) is conjugated toan anti-MUC16 antibody or antigen-binding fragment thereof via a linker,as shown in Formula (IIA), below:

wherein:

-   -   BA is an anti-MUC16 antibody or antigen-binding fragment        thereof;    -   a is an integer from 1 to 30;    -   Z₂ is represented by the following structural formula:        —Z_(2A)-Z_(2B)—Z_(2C)—Z_(2D), wherein Z_(2A), Z_(2B), Z_(2C) and        Z_(2D) are each independently absent, an amino acid, a peptide        having 2-20 amino acids, an alkyl, an alkynyl, an alkenyl, a        cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, —CR₅R₆—, —O—,        —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,        —C(═O)—(CH_(x))_(p1), —C(═O)—O—(CH_(x))_(p1),        —(CH₂)_(p1)—C(═O)—O—, —(O—(CH₂)_(p2)—)_(p3)—,        —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—, —C(═S)—S—, —C(═S)—NH—,        —S—C(═S)—, —S—C(═S)—S—, —S—, —SO—, —SO₂—, —NR₄—,        —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,        —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, —O—C(═O)—N(R₄),        —O—C(═S)—N(R₄)—, —C(═S)—N(R₄)—, —N═C═S, —N═C═O,

-   -   A is a natural or non-natural amino acid, or a peptide        comprising 2-20 amino acids;    -   W is —O—, —S—, —CR₅R₆—, or —NR₄—;    -   X is aryl, heteroaryl, cycloalkyl, or heterocyclyl, wherein        aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionally        substituted;    -   wherein A₁, A₃, and R₁ are each independently an amino acid, a        peptide having 2-20 amino acids, an alkyl, an alkynyl, an        alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl,        —CR₅R₆—, —O—, —C(═O)—, —O—C(═O)—, —C(═O)—O—, —O—C(═O)—O—,        —C(═O)—(CH_(x))_(p1)—, —C(═O)—O—(CH_(x))_(p1)—,        —(CH_(x))_(p1)—C(═O)—, —(CH_(x))_(p1)—C(═O)—O—,        —(O—(CH₂)_(p2)—)_(p3)—, —((CH₂)_(p2)—O—)_(p3)—, —C(═S)—,        —C(═S)—S—, —S—C(═S)—, —C(═S)—NH—, —S—C(═S)—S—, —S—, —SO—, —SO₂—,        —NR₄—, —N(R₄)—C(═O)—N(R₈)—, —N(R₄)—C(═O)O—, —N(R₄)—C(═O)—,        —C(═O)—N(R₄)—, —C(═O)—N(R₄)—C(═O)—, or —O—C(═O)—NR₄—, wherein        alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, and        heterocyclyl are optionally substituted;    -   R₁₇ is selected from the group consisting of O, S, NR₁₈, and        CR₅R₆;    -   R₁₈ is selected from the group consisting of H, alkyl, alkynyl,        alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and acyl,        wherein alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl,        heterocyclyl, and acyl are optionally substituted;    -   R₄, R₅, R₆ and R₈ are each independently H, or a substituted or        unsubstituted: alkyl, alkenyl, alkynyl, aryl, heteroaryl, or        heterocyclyl;    -   R_(4a) is a substituted or unsubstituted: alkyl, alkenyl,        alkynyl, aryl, heteroaryl, or heterocyclyl;    -   p1, p2 and p3 are each independently 0, or an integer from 1 to        100; and    -   x is 0, 1 or 2.

In some embodiments of Formula (IIA), A is a peptide selected from thegroup consisting of valine-citrulline, citrulline-valine,lysine-phenylalanine, phenylalanine-lysine, valine-asparagine,asparagine-valine, threonine-asparagine, asparagine-threonine,serine-asparagine, asparagine-serine, phenylalanine-asparagine,asparagine-phenylalanine, leucine-asparagine, asparagine-leucine,isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine,asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid,citrulline-asparagine, asparagine-citrulline, alanine-asparagine, andasparagine-alanine.

In one embodiment, the compound of Formula (IIA) that is bound to theanti-MUC16 antibody or antigen-binding fragment thereof is:

wherein

is a bond to the anti-MUC16 antibody or fragment thereof. In someinstances, this moiety is referred to as “Compound 7.”

In some embodiments, the cytotoxic agent that is conjugated to ananti-MUC16 antibody or fragment thereof is a pure, or substantiallypure, diastereomer of DM1:

and y is an integer 1 to 0.

In another embodiment, the ADC comprises a “A-[L-P]_(y)” structure inwhich A is an anti-MUC16 antibody or antigen-binding fragment thereof,and [L-P] is:

or

-   -   a mixture thereof, and    -   wherein y is an integer 1 to 30, and

is a bond to the anti-MUC16 antibody or fragment thereof.

Other maytansinoid derivatives are discussed in WO 2014/145090,WO2016/160615, and WO 2015/031396, each of which is hereby incorporatedby reference in its entirety.

In some embodiments, the cytotoxic agent that is conjugated to ananti-MUC16 antibody or fragment thereof is MMAE or MMAF.

Other cytotoxic agents known in the art are contemplated within thescope of the present invention, including, e.g., protein toxins such asricin, C. difficile toxin, pseudomonas exotoxin, diphtheria toxin,botulinum toxin, bryodin, saporin, pokeweed toxins (i.e.,phytolaccatoxin and phytolaccigenin), and others such as those set forthin Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

Cytotoxic agents (“payloads”) can be tethered to an anti-MUC16antigen-binding molecule or antibody of the invention via a chemicallinker that covalently binds the payload compound to the proteinmolecule (i.e. antibody). Exemplary embodiments of specific linkers arediscussed above. More generally, and as used herein, the term “linker”refers to any divalent group or moiety that links, connects, or bonds abinding agent (e.g., an antibody or an antigen-binding fragment thereof)with a payload compound set forth herein. Generally, suitable bindingagent linkers for the antibody conjugates described herein are thosethat are sufficiently stable to exploit the circulating half-life of theantibody and, at the same time, capable of releasing its payload afterantigen-mediated internalization of the conjugate. Linkers can becleavable or non-cleavable. Cleavable linkers are linkers that arecleaved by intracellular metabolism following internalization, e.g.,cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavablelinkers are linkers that release an attached payload via lysosomaldegradation of the antibody following internalization. Suitable linkersinclude, but are not limited to, acid-labile linkers, hydrolysis-labilelinkers, enzymatically cleavable linkers, reduction labile linkers,self-immolative linkers, and non-cleavable linkers. Suitable linkersalso include, but are not limited to, those that are or comprisepeptides, glucuronides, succinimide-thioethers, polyethylene glycol(PEG) units, hydrazones, mal-caproyl units, dipeptide units,valine-citrulline units, and para-aminobenzyl (PAB) units. In somecases, the linker is capable of bonding to the antibody orantigen-binding fragment through a lysine residue or a cysteine residue(e.g., via cleavage of a disulfide group of the antibody or fragment, orvia a cysteine residue engineered into the antibody or fragment). Insome cases, the linker is capable of bonding to the antibody or fragmentthrough a glutamine residue, including those derived viatransglutaminase-mediated conjugation.

Exemplary linkers that can be used in the context of the presentinvention include linkers that comprise or consist of e.g., MC(6-maleimidocaproyl), MCC (maleimidomethyl cyclohexane-1-carboxylate),MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala(valine-alanine), ala-phe (alanine-phenylalanine), phe-lys(phenylalanine-lysine), dipeptide site in protease-cleavable linker, PAB(p-aminobenzyloxycarbonyl), SPP (N-Succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate), SIAB (N-Succinimidyl (4-iodo-acetyl)aminobenzoate), andvariants and combinations thereof. Additional examples of linkers thatcan be used in the context of the present invention are disclosed in,e.g., U.S. Pat. No. 7,754,681 and in Ducry, Bioconjugate Chem., 2010,21:5-13, and the references cited therein, the contents of which areincorporated by reference herein in their entireties. In some cases, thelinker is or contains a self-immolative spacer, such as those discussedin Jin, et al., Bioorganic & Medicinal Chemistry, 2012, 20:3465-3469,and Wu, et al., Bioorganic & Medicinal Chemistry, 2016, 24:2697-2706.

Payloads may be linked to the anti-MUC16 antibody or antigen-bindingfragment via an attachment at a particular amino acid within theantibody or antigen-binding molecule. Exemplary amino acid attachmentsthat can be used in the context of this aspect of the invention include,e.g., lysine (see, e.g., U.S. Pat. No. 5,208,020; US 2010/0129314;Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808;U.S. Pat. No. 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine(see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and U.S.Pat. No. 7,750,116), selenocysteine (see, e.g., WO 2008/122039; andHofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456),formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007,3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51,and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-naturalamino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidicamino acids (see, e.g., WO 2012/05982). Linkers can also be conjugatedto an antigen-binding protein via attachment to carbohydrates (see,e.g., US 2008/0305497, and Ryan et al., Food & Agriculture Immunol.,2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO2010/010324, WO 2011/018611, and Shaunak et al., Nat. Chem. Biol., 2006,2:312-313).

Drug-to-antibody ratio (DAR) is the average number of drugs conjugatedto the antibody or antigen-binding fragment, which has an importanteffect on the efficacy, potency and pharmacokinetics of the ADC. Invarious embodiments, the DAR is from 1, 2, 3, 4, 5, 6, 7, or 8 drugmolecules per antibody. In some embodiments, the DAR is from 1 to 4. Incertain embodiments, the DAR is from 2 to 4. In some cases, the DAR isfrom 2 to 3. In certain cases, the DAR is from 3 to 4. In someembodiments, the DAR is from 1 to 10, 1 to 20 or 1 to 30 (i.e., from 1to 30 drug molecules per antibody or antigen-binding fragment thereof).

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe antigen-binding molecules of the present invention. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area. Whena bispecific antigen-binding molecule of the present invention is usedfor therapeutic purposes in an adult patient, it may be advantageous tointravenously administer the bispecific antigen-binding molecule of thepresent invention normally at a single dose of about 0.01 to about 20mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 toabout 5, or about 0.05 to about 3 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering a bispecific antigen-binding molecule may be determinedempirically; for example, patient progress can be monitored by periodicassessment, and the dose adjusted accordingly. Moreover, interspeciesscaling of dosages can be performed using well-known methods in the art(e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-MUC16 antibody or antigen-binding fragment thereof, or a bispecificantigen-binding molecule that specifically binds CD3 and MUC16. Thetherapeutic composition can comprise any of the antibodies or bispecificantigen-binding molecules as disclosed herein and a pharmaceuticallyacceptable carrier or diluent. As used herein, the expression “a subjectin need thereof” means a human or non-human animal that exhibits one ormore symptoms or indicia of cancer (e.g., a subject expressing a tumoror suffering from any of the cancers mentioned herein below), or whootherwise would benefit from an inhibition or reduction in MUC16activity or a depletion of MUC16+ cells (e.g., ovarian cancer cells).

The antibodies and bispecific antigen-binding molecules of the invention(and therapeutic compositions comprising the same) are useful, interalia, for treating any disease or disorder in which stimulation,activation and/or targeting of an immune response would be beneficial.In particular, the anti-MUC16 antibodies or the anti-CD3/anti-MUC16bispecific antigen-binding molecules of the present invention may beused for the treatment, prevention and/or amelioration of any disease ordisorder associated with or mediated by MUC16 expression or activity orthe proliferation of MUC16+ cells. The mechanism of action by which thetherapeutic methods of the invention are achieved include killing of thecells expressing MUC16 in the presence of effector cells, for example,by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two ormore of these mechanisms. Cells expressing MUC16 which can be inhibitedor killed using the bispecific antigen-binding molecules of theinvention include, for example, ovarian cancer cells.

The antigen-binding molecules of the present invention may be used totreat a disease or disorder associates with MUC16 expression including,e.g., a cancer including ovarian cancer, breast cancer, pancreaticcancer, non-small-cell lung cancer, intrahepatic cholangiocarcinoma-massforming type, adenocarcinoma of the uterine cervix, and adenocarcinomaof the gastric tract. According to certain embodiments of the presentinvention, the anti-MUC16 antibodies or anti-MUC16/anti-CD3 bispecificantibodies are useful for treating a patient afflicted with ovariancancer. According to other related embodiments of the invention, methodsare provided comprising administering an anti-MUC16 antibody or ananti-CD3/anti-MUC16 bispecific antigen-binding molecule as disclosedherein to a patient who is afflicted with ovarian cancer.Analytic/diagnostic methods known in the art, such as tumor scanning,etc., may be used to ascertain whether a patient harbors an ovariantumor.

The present invention also includes methods for treating residual cancerin a subject. As used herein, the term “residual cancer” means theexistence or persistence of one or more cancerous cells in a subjectfollowing treatment with an anti-cancer therapy.

According to certain aspects, the present invention provides methods fortreating a disease or disorder associated with MUC16 expression (e.g.,ovarian cancer) comprising administering one or more of the anti-MUC16or bispecific antigen-binding molecules described elsewhere herein to asubject after the subject has been determined to have prostate cancer.For example, the present invention includes methods for treating ovariancancer comprising administering an anti-MUC16 antibody or ananti-CD3/anti-MUC16 bispecific antigen-binding molecule to a patient 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more afterthe subject has received hormone therapy (e.g., anti-androgen therapy).

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary antibodiesand bispecific antigen-binding molecules described herein in combinationwith one or more additional therapeutic agents. Exemplary additionaltherapeutic agents that may be combined with or administered incombination with an antigen-binding molecule of the present inventioninclude, e.g., an EGFR antagonist (e.g., an anti-EGFR antibody [e.g.,cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g.,gefitinib or erlotinib]), an antagonist of another EGFR family membersuch as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 oranti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4activity), an antagonist of EGFRvIII (e.g., an antibody thatspecifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMETantibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-rafinhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-αinhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g.,an anti-PDGFR-β antibody), a VEGF antagonist (e.g., a VEGF-Trap, see,e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a“VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), a FOLH1 (PSMA) antagonist, a PRLRantagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist(e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., ananti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), auroplakin antagonist (e.g., an anti-uroplakin antibody), etc. Otheragents that may be beneficially administered in combination with theantigen-binding molecules of the invention include cytokine inhibitors,including small-molecule cytokine inhibitors and antibodies that bind tocytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11,IL-12, IL-13, IL-17, IL-18, or to their respective receptors. Thepharmaceutical compositions of the present invention (e.g.,pharmaceutical compositions comprising an anti-CD3/anti-MUC16 bispecificantigen-binding molecule as disclosed herein) may also be administeredas part of a therapeutic regimen comprising one or more therapeuticcombinations selected from “ICE”: ifosfamide (e.g., Ifex®), carboplatin(e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®,VP-16); “DHAP”: dexamethasone (e.g., Decadron®), cytarabine (e.g.,Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g.,Platinol®-AQ); and “ESHAP”: etoposide (e.g., Etopophos®, Toposar®,VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dosecytarabine, cisplatin (e.g., Platinol®-AQ).

The present invention also includes therapeutic combinations comprisingany of the antigen-binding molecules mentioned herein and an inhibitorof one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII,cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1,STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementionedcytokines, wherein the inhibitor is an aptamer, an antisense molecule, aribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment(e.g., Fab fragment; F(ab′)2 fragment; Fd fragment; Fv fragment; scFv;dAb fragment; or other engineered molecules, such as diabodies,triabodies, tetrabodies, minibodies and minimal recognition units). Theantigen-binding molecules of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids and/or NSAIDs. The antigen-binding moleculesof the invention may also be administered as part of a treatment regimenthat also includes radiation treatment and/or conventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan antigen-binding molecule of the present invention; (for purposes ofthe present disclosure, such administration regimens are considered theadministration of an antigen-binding molecule “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which anantigen-binding molecule of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an antigen-binding molecule (e.g., an anti-MUC16 antibody or abispecific antigen-binding molecule that specifically binds MUC16 andCD3) may be administered to a subject over a defined time course. Themethods according to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an antigen-binding moleculeof the invention. As used herein, “sequentially administering” meansthat each dose of an antigen-binding molecule is administered to thesubject at a different point in time, e.g., on different days separatedby a predetermined interval (e.g., hours, days, weeks or months). Thepresent invention includes methods which comprise sequentiallyadministering to the patient a single initial dose of an antigen-bindingmolecule, followed by one or more secondary doses of the antigen-bindingmolecule, and optionally followed by one or more tertiary doses of theantigen-binding molecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of an antigen-binding molecule contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule (e.g., an anti-MUC16 antibody or abispecific antigen-binding molecule that specifically binds MUC16 andCD3). For example, in certain embodiments, only a single secondary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to thepatient. Likewise, in certain embodiments, only a single tertiary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to thepatient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-MUC16 antibodies of the present invention may also be used todetect and/or measure MUC16, or MUC16-expressing cells in a sample,e.g., a biological sample for diagnostic purposes. For example, ananti-MUC16 antibody, or fragment thereof, may be used to diagnose acondition or disease characterized by aberrant expression (e.g.,over-expression, under-expression, lack of expression, etc.) of MUC16.Exemplary diagnostic assays for MUC16 may comprise, e.g., contacting asample, obtained from a patient, with an anti-MUC16 antibody of theinvention, wherein the anti-MUC16 antibody is labeled with a detectablelabel or reporter molecule. Alternatively, an unlabeled anti-MUC16antibody can be used in diagnostic applications in combination with asecondary antibody which is itself detectably labeled. The detectablelabel or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P,³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, beta-galactosidase, horseradish peroxidase, or luciferase.Another exemplary diagnostic use of the anti-MUC16 antibodies of theinvention includes ⁸⁹Zr-labeled, such as ⁸⁹Zr-desferrioxamine-labeled,antibody for the purpose of noninvasive identification and tracking oftumor cells in a subject (e.g. positron emission tomography (PET)imaging). (See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan. 1;76(1):73-82; and Azad, B B. et al. Oncotarget. 2016 Mar. 15;7(11):12344-58.) Specific exemplary assays that can be used to detect ormeasure MUC16 in a sample include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting(FACS).

Samples that can be used in MUC16 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of MUC16 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of MUC16 in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal MUC16 levels or activity) will be measured to initiallyestablish a baseline, or standard, level of MUC16. This baseline levelof MUC16 can then be compared against the levels of MUC16 measured insamples obtained from individuals suspected of having a MUC16 relateddisease (e.g., a tumor containing MUC16-expressing cells) or condition.Examples of tissue or fluid samples include, but are not limited toplasma, serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas,stomach, small or large intestine, gall bladder, breast, lung, kidney,salivary, and lacrimal glands, or any epithelioid malignancy thereof.Additional examples of tissue or fluid samples include, but are notlimited to papillary serous carcinoma of the cervix, adenocarcinoma ofthe endometrium, clear cell adenocarcinoma of the bladder, seminalvesicle carcinoma, gastric carcinoma, colorectal adenocarcinoma andepithelioid mesothelioma. It is envisioned that any fluid or tissuesample which contains detectable quantities of MUC16 protein, orfragments thereof, may be subjected to the detection methods describedherein. The described methods may be used to monitor the development andprogression of malignant diseases, or to distinguish between normal anddisease conditions. As such, the described methods may be used to detector monitor cancers, such as ovarian cancer, bladder cancer, breastcancer, pancreatic cancer, non-small-cell lung cancer, intrahepaticcholangiocarcinoma-mass forming type, adenocarcinoma of the uterinecervix, and adenocarcinoma of the gastric tract.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Generation of Anti-MUC16 Antibodies

Anti-MUC16 antibodies were obtained by immunizing a genetically modifiedmouse with a human MUC16 antigen or by immunizing an engineered mousecomprising DNA encoding human immunoglobulin heavy and kappa light chainvariable regions with a human MUC16 antigen.

Genetically modified mice were immunized with hMUC16.nub (a truncatedformat encompassing the last five SEA domains of Mucin-16 (SEQ ID:1902)), or immunized with an hMUC16-expressing cell line, such asOVCAR-3 cells. SEQ ID NO: 1902 contains residues 13810-14451 of SEQ IDNO: 1899, as well as C-terminal tags. Following immunization,splenocytes were harvested from each mouse and either (1) fused withmouse myeloma cells to preserve their viability and form hybridoma cellsand screened for MUC16 specificity, or (2) B-cell sorted (as describedin US 2007/0280945A1) using a human MUC16 fragment as the sortingreagent that binds and identifies reactive antibodies (antigen-positiveB cells).

Chimeric antibodies to MUC16 were initially isolated having a humanvariable region and a mouse constant region. The antibodies werecharacterized and selected for desirable characteristics, includingaffinity, selectivity, etc. If necessary, mouse constant regions werereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4 constant region, to generate a fully humananti-MUC16 antibody. While the constant region selected may varyaccording to specific use, high affinity antigen-binding and targetspecificity characteristics reside in the variable region. The antibodyname designations such as H1H8755P and H1M7129N denote fully humanantibodies “H1H” or chimeric human variable/mouse constant regionantibodies “HIM”. Antibodies identified by the hybridoma method areindicated with antibody ID numbers ending with “N” or “N2.” Antibodiesidentified by the B-cell sorting method are indicated with antibody IDnumbers ending with “P” or “P2”.

Certain biological properties of the exemplary anti-MUC16 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Heavy and Light Chain Variable Region Amino Acid and Nucleic AcidSequences of Anti-MUC16 Antibodies

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-MUC16 antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H8755P 2 4 6 8 10 12 1416 H1H8767P 18 20 22 24 26 28 30 32 H1H8770P 34 36 38 40 42 44 46 48H1H8783P 50 52 54 56 58 60 62 64 H1H8790P 66 68 70 72 74 76 78 80H1H8794P 82 84 86 88 90 92 94 96 H1H8794P2 82 84 86 88 858 860 862 864H1H8799P 98 100 102 104 106 108 110 112 H1H8799P2 98 100 102 104 170 172174 176 H1H8804P 114 116 118 120 122 124 126 128 H1H8808P 130 132 134136 138 140 142 144 H1H8810P 146 148 150 152 154 156 158 160 H1H8813P162 164 166 168 170 172 174 176 H1M7129N 178 180 182 184 186 188 190 192H1M7137N 194 196 198 200 394 396 398 400 H1M9519N 202 204 206 208 210212 214 216 H1M9521N 218 220 222 224 226 228 230 232 H1M9528N 234 236238 240 242 244 246 248 H2M7128N 250 252 254 256 1936 1938 1940 1942H1M7130N 1944 1946 1948 1950 1952 1954 1956 1958 H2M7131N 258 260 262264 266 268 270 272 H2M7133N 274 276 278 280 1936 1938 1940 1942H2M7134N 282 284 286 288 290 292 294 296 H2M7135N 298 300 302 304 306308 310 312 H2M7138N 314 316 318 320 322 324 326 328 H2M9538N 330 332334 336 338 340 342 344 H3M9524N 346 348 350 352 354 356 358 360H3M9525N 362 364 366 368 370 372 374 376 H3M9529N 378 380 382 384 386388 390 392

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H8755P 1 3 57 9 11 13 15 H1H8767P 17 19 21 23 25 27 29 31 H1H8770P 33 35 37 39 41 4345 47 H1H8783P 49 51 53 55 57 59 61 63 H1H8790P 65 67 69 71 73 75 77 79H1H8794P 81 83 85 87 89 91 93 95 H1H8794P2 81 83 85 87 857 859 861 863H1H8799P 97 99 101 103 105 107 109 111 H1H8799P2 97 99 101 103 169 171173 175 H1H8804P 113 115 117 119 121 123 125 127 H1H8808P 129 131 133135 137 139 141 143 H1H8810P 145 147 149 151 153 155 157 159 H1H8813P161 163 165 167 169 171 173 175 H1M7129N 177 179 181 183 185 187 189 191H1M7137N 193 195 197 199 393 395 397 399 H1M9519N 201 203 205 207 209211 213 215 H1M9521N 217 219 221 223 225 227 229 231 H1M9528N 233 235237 239 241 243 245 247 H2M7128N 249 251 253 255 1935 1937 1939 1941H1M7130N 1943 1945 1947 1949 1951 1953 1955 1957 H2M7131N 257 259 261263 265 267 269 271 H2M7133N 273 275 277 279 1935 1937 1939 1941H2M7134N 281 283 285 287 289 291 293 295 H2M7135N 297 299 301 303 305307 309 311 H2M7138N 313 315 317 319 321 323 325 327 H2M9538N 329 331333 335 337 339 341 343 H3M9524N 345 347 349 351 353 355 357 359H3M9525N 361 363 365 367 369 371 373 375 H3M9529N 377 379 381 383 385387 389 391

Example 2: Anti-MUC16 Antibodies Bind Specifically to EndogenouslyExpressed hMUC16 on OVCAR-3 Cell Line

The ability of anti-MUC16 antibodies to bind specifically toendogenously expressing MUC16 on the human ovarian carcinoma cell line(OVCAR-3) was evaluated via an electrochemiluminescence based detectionassay (Meso Scale Discovery (MSD), Rockville, Md.). Briefly, OVCAR-3 anda control ovarian adenocarcinoma cell line, SK-OV-3, which has nodetectable hMUC16 expression, were rinsed in 1×PBS supplemented withCa²⁺/Mg²⁺ (Irvine Scientific, Santa Ana, Calif.) followed by incubationin Enzyme Free Cell Dissociation buffer (Millipore, Billerica, Mass.)for 10 min at 37° C. Detached cells were then washed once in 1×PBSsupplemented with Ca²⁺/Mg²⁺ and counted (Cellometer Auto T4 cellcounter, Nexcelom Bioscience, Lawrence, Mass.). Approximately 1.0×10⁴cells were plated in MULTI-ARRAY 96-well carbon electrode plates (MSD)and incubated for 1 h at 37° C. Nonspecific binding sites were thenblocked by 2% BSA (w/v) in PBS for 1 h at room temperature. Next, serialdilutions of anti-MUC16 or control antibodies (0.85 pM-50 nM) andno-antibody buffer controls were added to the plate-bound cells andincubated for 1 h at room temperature (RT). Plates were then washed toremove the unbound antibodies using an AquaMax2000 plate washer (MDSAnalytical Technologies, Sunnyvale, Calif.). Plate-bound antibodies weredetected with a SULFO-TAG™-conjugated anti-human kappa light chainantibody (Regeneron) or a SULFO-TAG™-conjugated anti-mouse IgG antibody(Jackson Immunoresearch, West Grove, Pa.) for 1 h at RT. Post-wash,plates were developed with Read Buffer (MSD) according to manufacturer'sprotocol and luminescent signals were recorded with a SECTOR Imager 6000(MSD) instrument.

Luminescence intensity, measured in relative light units (RLU), for thetwo cell lines was recorded to indicate the binding intensity of eachantibody. The ratio of signal detected with 1.9 nM or 16.7 nM anti-MUC16antibody binding to OVCAR-3 vs. SK-OV-3 was reported as an indication ofspecificity and potency of binding (Table 3).

TABLE 3 Cell Binding Ratios of Anti-Muc16 Antibodies to EndogenouslyExpressing hMUC16 OVCAR-3 vs hMUC16-negative SK-OV-3 Cell Lines Ratio:anti-MUC16 Binding OVCAR-3/SK-OV-3 Antibody ID [Ab]: 1.9 nM [Ab]: 16.7nM Hybridoma Anti-Muc16 Antibodies (H1M, H2M, H3M) H2M7128N 59 31H1M7129N 68 19 H2M7131N 86 37 H2M7133N 69 33 H2M7134N 68 29 H2M7135N 305 H1M7137N 77 29 H2M7138N 82 50 H3M7132N 89 38 H1M9519N 109 78 H1M9521N132 107 H3M9524N 81 57 H3M9525N 137 51 H1M9528N 153 120 H3M9529N 143 99H2M9538N 89 26 Human Fc anti-MUC16 Antibodies (H1H) H1H8755P 13 5H1H8767P 4 NS H1H8770P 9 3 H1H8783P 11 4 H1H8790P 8 3 H1H8794P 8 3H1H8794P2 5 NS H1H8799P 10 4 H1H8799P2 10 4 H1H8804P 8 3 H1H8808P 4 NSH1H8810P 8 3 H1H8813P 11 5 H1H9519N 41 30 H1H9521N 30 28 H1H9524N 20 48H1H9525N 4 21 H1H9528N 41 18 H1H9529N 109 74 H1H9538N 12 13 ControlsmIgG1 Isotype Control NS NS Antibody mIgG2a Isotype NS NS ControlAntibody hIgG1 Isotype Control NS NS Antibody NS = Non-Specific-ratio at1.9 nM or 16.7 nM < 2.5-fold. Isotypes: H1H: hIgG1; H1M: mIgG1; H2M:mIgG2; H3M: mIgG3 Note: variation in binding intensity ratios betweenthe same antibody expressed with a mouse Fc and human Fc is due to theuse of different SULFO-TAG secondary detect reagents.

As the results in Table 3 show, a majority of the anti-MUC16 antibodiesof this invention bound specifically to OVCAR-3 at both high (16.7 nM)and low (1.9 nM) antibody concentrations. mIgG1, mIgG2a and hIgG1Isotype Control antibodies showed no specific binding to either OVCAR-3or the SK-OV-3 cell line. Additionally, evidence exists that the methodis sensitive since several antibodies that did not exhibit binding tosoluble monomeric human MUC16 protein in a surface plasmon resonancebinding assay (see Example 4 hereinbelow) displayed specific binding toendogenous human MUC16 expressed on OVCAR-3 cells in this cell-basedbinding assay.

Example 3: Generation of Bispecific Antibodies that Bind OvarianCell-Specific (MUC16) and CD3

The present invention provides bispecific antigen-binding molecules thatbind CD3 and MUC16; such bispecific antigen-binding molecules are alsoreferred to herein as “anti-MUC16/anti-CD3 or anti-MUC16×CD3 bispecificmolecules.” The anti-MUC16 portion of the anti-MUC16/anti-CD3 bispecificmolecule is useful for targeting tumor cells that express MUC16 (alsoknown as CA-125), and the anti-CD3 portion of the bispecific molecule isuseful for activating T-cells. The simultaneous binding of MUC16 on atumor cell and CD3 on a T-cell facilitates directed killing (cell lysis)of the targeted tumor cell by the activated T-cell.

Bispecific antibodies comprising an anti-MUC16-specific binding domainand an anti-CD3-specific binding domain were constructed using standardmethodologies, wherein the anti-MUC16 antigen binding domain and theanti-CD3 antigen binding domain each comprise different, distinct HCVRspaired with a common LCVR. In exemplified bispecific antibodies, themolecules were constructed utilizing a heavy chain from an anti-CD3antibody, a heavy chain from an anti-MUC16 antibody and a common lightchain from the anti-MUC16 antibody. In other instances, the bispecificantibodies may be constructed utilizing a heavy chain from an anti-CD3antibody, a heavy chain from an anti-MUC16 antibody and a light chainfrom an anti-CD3 antibody or an antibody light chain known to bepromiscuous or pair effectively with a variety of heavy chain arms.

The bispecific antibodies described in the following examples consist ofanti-CD3 binding arms having varying binding affinities to human solubleheterodimeric hCD3ε/δ protein (as described in Example 15 herein); andhuman MUC16 (see Examples 1-2 above). Exemplified bispecific antibodieswere manufactured having an IgG1 Fc domain (BSMUC16/CD3-001, -002, -003,and -004) or a modified (chimeric) IgG4 Fc domain (BSMUC16/CD3-005) asset forth in US Patent Application Publication No. US20140243504A1,published on Aug. 28, 2014.

A summary of the component parts of the antigen-binding domains of thevarious anti-MUC16×CD3 bispecific antibodies constructed is set forth inTable 4.

TABLE 4 Summary of Component Parts of Selected Anti-MUC16 × CD3Bispecific Antibodies Anti-MUC16 Anti-CD3 Antigen-BindingAntigen-Binding Bispecific Domain Domain Common Antibody Heavy ChainHeavy Chain Light Chain Identifier Variable Region Variable RegionVariable Region BSMUC16/ H1H8767P CD3-VH-G H1H8767P CD3-001 (SEQ ID NO:18) (SEQ ID (SEQ ID NO: 26) NO: 1730) BSMUC16/ H1H8767P CD3-VH-G5H1H8767P CD3-002 (SEQ ID NO: 18) (SEQ ID (SEQ ID NO: 26) NO: 1762)BSMUC16/ H1H8767P CD3-VH-G9 H1H8767P CD3-003 (SEQ ID NO: 18) (SEQ ID(SEQ ID NO: 26) NO: 1778) BSMUC16/ H1H8767P CD3-VH-G10 H1H8767P CD3-004(SEQ ID NO: 18) (SEQ ID (SEQ ID NO: 26) NO: 1786) BSMUC16/ H1H8767PCD3-VH-G20 H1H8767P CD3-005 (SEQ ID NO: 18) (SEQ ID (SEQ ID NO: 26) NO:1866)

The light chains listed in Table 4 were common to both the CD3 and MUC16targeting arms of the bispecific antibodies. Tables 1 and 2 set outamino acid and nucleic acid sequence identifiers, respectively, for thevarious heavy chain variable regions, and their corresponding CDRs, ofthe anti-MUC16 arms of the bispecific antibodies of this Example. Table22 and 23 set out amino acid and nucleic acid sequence identifiers,respectively, for the various heavy chain variable regions, and theircorresponding CDRs, of the anti-CD3 arms of the bispecific antibodies ofthis Example.

Example 4: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-MUC16 Monospecific andAnti-MUC16×CD3 Bispecific Antibodies

Binding affinities and kinetic constants of human anti-MUC16 antibodieswere determined via real-time surface plasmon resonance (SPR; Biacore4000 or Biacore T-200, GE Healthcare Life Sciences, Pittsburgh, Pa.) at25° C. The anti-MUC16 antibodies tested in this example were bivalentmonospecific binders to MUC16 (expressed with a hIgG1 (H1H), mIgG1(HIM), mIgG2 (H2M) or mIgG3 (H3M) constant region) or bispecificantibodies comprised of an anti-MUC16 binding domain and an anti-CD3binding domain. Antibodies were captured onto a CM4 or CM5 Biacoresensor surface (GE Healthcare Life Sciences) derivatized via aminecoupling with a monoclonal anti-human Fc antibody (GE, #BR-1008-39) or amonoclonal goat anti-mouse Fc antibody (GE, #BR-1008-38). Variousconcentrations of soluble monomeric human MUC16, in a truncated formatencompassing the last five SEA domains of Mucin-16 (hMUC16.mmh, or MUC16“nub”, SEQ ID: 1902) were injected over the anti-MUC16-antibody capturedsurface at a flow rate of 50 uL/minute (Biacore T-200) or 30 uL/minute(Biacore 4000). Antibody-reagent association was monitored for 4 min andthe dissociation was monitored for 6-10 min. All binding studies wereperformed in HBS-ET buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% v/vSurfactant P20).

Kinetic association (k_(a)) and dissociation (k_(d)) rate constants weredetermined by fitting the real-time sensorgrams to a 1:1 binding modelusing Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as:

${{K_{D}(M)} = \frac{kd}{ka}},{{{and}{t^{1/2}\left( \min \right)}} = \frac{\ln(2)}{60*{kd}}}$

Binding kinetic parameters for the monospecific anti-MUC16 antibodies toa monomeric human MUC16 protein fragment are shown below in Tables 5Aand 5B. Binding kinetic parameters for the anti-MUC16/anti-CD3bispecific antibodies to monomeric human MUC16 protein are shown belowin Table 6.

TABLE 5A Biacore binding affinities of Hybridoma Anti-MUC16 antibodies(H1M, H2M and H3M) to hMUC16 fragment at 25° C. Antibody t ½ ID ka(1/Ms) kd (1/s) KD (M) (min) H2M7128N 1.89E+05 6.40E−04 3.39E−09 18H1M7129N 5.31E+04 1.04E−04 1.97E−09 111 H2M7131N 6.47E+04 1.62E−042.51E−09 71 H3M7132N 2.57E+04 1.96E−04 7.62E−09 59 H2M7133N 1.67E+053.77E−04 2.26E−09 31 H2M7134N 6.55E+04 1.62E−04 2.47E−09 71 H2M7135N5.10E+04 2.18E−04 4.27E−09 53 H1M7137N 5.30E+04 9.09E−05 1.72E−09 127H2M7138N 7.41E+04 9.25E−05 1.25E−09 125 H1M9519N NB NB NB NB H1M9521N NBNB NB NB H3M9524N NB NB NB NB H3M9525N NB NB NB NB H1M9528N NB NB NB NBH3M9529N NB NB NB NB NB: No binding

TABLE 5B Biacore binding affinities of Human Fc anti-MUC16 antibodies(H1H) to hMUC16 fragment at 25° C. Antibody t_(1/2) ID ka (1/Ms) kd(1/s) KD (M) (min) H1H8755P 5.22E+05 1.49E−04 2.86E−10 77 H1H8767P1.17E+05 4.18E−04 3.58E−09 28 H1H8770P 2.47E+05 3.08E−04 1.25E−09 38H1H8783P 1.74E+05 1.07E−04 6.14E−10 108 H1H8790P 1.01E+05 7.61E−047.53E−09 15 H1H8794P 3.62E+05 2.79E−04 7.71E−10 41 H1H8799P 7.90E+043.66E−04 4.63E−09 32 H1H8799P2 7.58E+04 3.73E−04 4.92E−09 31 H1H8804P4.94E+04 6.07E−04 1.23E−08 19 H1H8808P 4.12E+03 2.16E−04 5.24E−08 54H1H8810P 5.77E+04 3.16E−04 5.48E−09 37 H1H8813P 5.32E+04 2.32E−044.35E−09 50

TABLE 6 Biacore binding affinities of anti-MUC16/anti-CD3 BispecificAntibodies to hMUC16 fragment at 25° C. Bispecific Antibody t ½Identifier ka (1/Ms) kd (1/s) KD (M) (min) BSMUC16/CD3-001 9.48E+045.86E−04 6.18E−09 20 BSMUC16/CD3-005 9.41E+04 5.64E−04 6.00E−09 21

As the results show, a majority of the anti-MUC16 antibodies of thisinvention bound to the soluble human MUC16 protein, some displayingsub-nanomolar affinity. Several antibodies (H1M9519N, H1M9521N,H3M9524N, H3M9525N, H1M9528N, H3M9529N) displayed no binding to thetruncated format encompassing the last five SEA domains via surfaceplasmon resonance, however, displayed specific binding to endogenoushuman MUC16 expressed on OVCAR-3 cells in a cell-based binding assay.Anti-MUC16×CD3 bispecific antibodies of this invention also bound tosoluble truncated human MUC16 protein exhibiting nanomolar affinity inthis assay.

Example 5: Additional Binding, T-Cell Activation and CytotoxicityProperties of Exemplified Bispecific Antibodies

In this example, the ability of MUC16×CD3 bispecific antibodies to bindto human CD3-expressing (human T cell) cell lines, compared to bindingto target-specific (MUC16-specific) cell lines, via FACS was determined.Additionally, the ability of these bispecific antibodies to activate totarget-specific (MUC16-specific) cell lines was also compared in asimilar assay.

Binding Titration of Exemplified Bispecific Antibodies as Measured byFACS Analysis

A. Briefly, flow cytometric analysis (i.e. fluorescence-activated cellsorting, or FACS) was utilized to determine binding of bispecificantibodies to Jurkat cells or cells expressing human MUC16, followed bydetection with a phycoerythrin (PE)-labeled or APC-labeled anti-humanIgG antibody. Briefly, 2×10⁵ cells/well were incubated for 30 minutes at4° C. with a serial dilution ranging from 1.33E-07M to 8.03E-12M of eachtest antibody or isotype control (antibody of same isotype that binds adifferent antigen with no cross-reactivity to MUC16 or CD3). Afterincubation, the cells were washed twice with cold PBS containing 1%filtered FBS and a PE-conjugated or APC-conjugated anti-human secondaryantibody was added to the cells and incubated for an additional 30minutes. Wells containing no antibody or secondary only were also usedas controls. After incubation, cells were washed, re-suspended in 200 μLcold PBS containing 1% filtered FBS and analyzed by flow cytometry on aBD FACS Canto II. See Table 7A.

B. In separate experiments with analogous conditions to that describedabove, flow cytometric analysis (or FACS) was utilized to determinebinding of select bispecific antibodies to Jurkat cells, PEO-1,OVCAR3-Luc and cynomolgus T cells. For the titration analysis, serialdilution of selected MUC16×CD3 bispecific antibodies, a first isotypecontrol antibody (a human chimeric IgG4 antibody that binds anirrelevant human antigen with no cross-reactivity to human or cynomolgusCD3) and a second isotype control antibody (a human chimeric IgG4antibody that binds an irrelevant human antigen with no cross-reactivityto human MUC16), ranging from 66.6 nM to 0.001 nM. See Table 7B andFIGS. 11A-11C.

For FACS analysis, cells were gated by forward scatter height vs.forward scatter area for single events selection, followed by side andforward scatters. The EC50 for cell binding titration was determinedusing PRISM™ software (GraphPad Software, Inc., La Jolla, Calif.).Values were calculated using 4-parameter non-linear regression analysis.(Liu, J., et al. 2005, Biotechnol Letters 27:1821-1827). The EC50 valuerepresents the concentration of the tested antibody where 50% of itsmaximal binding is observed.

TABLE 7A FACS Binding on CD3 and MUC16-Specific Cell lines FACS bindingtitration EC₅₀ [M] Bispecific Antibody Anti-CD3 OVCAR3 IdentifierBinding Arm Jurkat (MUC16+) BSMUC16/CD3-001 CD3-VH-G 3.21E−09 1.20E−09BSMUC16/CD3-002 CD3-VH-G5 Very weak 2.69E−09

TABLE 7B FACS Binding on CD3 and MUC16-Specific Cell lines FACS bindingtitration EC₅₀ [M] Bispecific Antibody Anti-CD3 PEO-1 OVCAR3-LucCynomolgus Identifier Binding Arm (MUC16+) (MUC16+) Jurkat T cellsBSMUC16/CD3-001 CD3-VH-G 3.02E−09 1.32E−09 6.44E−09 1.56E−08BSMUC16/CD3-002 CD3-VH-G5 3.13E−09 Not tested 3.01E−07 No BindingBSMUC16/CD3-005 CD3-VH-G20 2.63E−09 1.47E−09 6.26E−08 1.17E−06

As shown in Tables 7A and 7B, the CD3 binding arms of each MUC16bispecific antibody displayed a range of cell binding to human andmonkey CD3 expressing T cells (e.g. from 3.2 nM EC₅₀ to very weakbinding). BSMUC16/CD3-001 bispecific antibody showed high measurement ofbinding to CD3-expressing cells (i.e. <7 nM) while the BSMUC16/CD3-002bispecific antibody showed weak-to-no binding to human and monkeyCD3-expressing cells. Non-measurable binding, or no measurable binding,in the FACS assay or equivalent assay refers to a binding interactionbetween the antibody and its target antigen which is beyond thedetection limit of the assay (e.g. at or above 1 μM). The testedbispecific antibodies displayed similar cell binding on MUC16-expressingcell lines, confirming that bispecific pairing with individual CD3 armsexhibiting high or weak (or no measurable) interactions with CD3 did notaffect or diminish tumor target-specific binding (MUC16-specific bindingwas less than or equal to 3 nM (high binding) in these examples. Thefirst control antibody did not bind to CD3+ cells, and the secondcontrol antibody did not bind to MUC16+ cells. See also FIGS. 11A-11C.

Antibodies exhibiting weak-to-no detectable binding to human CD3 arestill considered advantageous for avidity-driven bispecific pairing, andwere further tested for cytotoxicity in in vitro (see below) and in vivoassays (Example 8).

T-Cell Activation and Tumor-Specific Cytotoxicity Exhibited byBispecific Antibodies as Measured In Vitro

A. The specific killing of MUC16-expressing tumor target cells in thepresence of CD3-based bispecific antibodies was monitored via flowcytometry. As reported previously, the bispecific antibodies displayeddifferential binding abilities to CD3 protein and CD3-expressing celllines (i.e. very weak or strong binding). These same bispecificantibodies were tested for the ability to induce naïve human T-cells tore-direct killing toward target-expressing cells.

Briefly, MUC16-expressing (OVCAR3) cell lines were labeled with 1 μM ofthe fluorescent tracking dye Violet Cell Tracker. After labeling, cellswere plated overnight at 37° C. Separately, human PBMCs were plated insupplemented RPMI media at 1×10⁶ cells/mL and incubated overnight at 37°C. in order to enrich for lymphocytes by depleting adherent macrophages,dendritic cells, and some monocytes. The next day, target cells wereco-incubated with adherent cell-depleted unstimulated PBMC(Effector/Target cell 4:1 ratio) and a serial dilution of relevantbispecific antibodies or Isotype control (concentration range: 66.7 nMto 0.25 pM) for 48 hours at 37° C. Cells were removed from cell cultureplates using an enzyme-free cell dissociation buffer, and analyzed byFACS. See results represented in Table 8A.

B. In analogous studies, MUC16-expressing (PEO-1 or OVCAR3-Luc) celllines were labeled, plated and incubated overnight as described. Serialdilutions of MUC16×CD3 bispecific antibodies or isotype control wereco-incubated. See results depicted in Tables 8B and 8C, and FIGS.12A-12B.

For FACS analysis, cells were stained with a dead/live far red celltracker (Invitrogen). 5×10⁵ counting beads were added to each wellimmediately before FACS analysis. 1×10⁴ beads were collected for eachsample. For the assessment of specificity of killing, cells were gatedon live Violet labeled populations. Percent of live population wasrecorded and used for the calculation of normalized survival.

T cell activation was assessed by incubating cells with directlyconjugated antibodies to CD2, CD69 and/or CD25, and by reporting thepercent of early activated (CD69+) T cells and/or late activated (CD25+)T cells out of total T cells (CD2+).

As the results in Tables 8A-8C show, depletion of MUC16-expressing cellswas observed with anti-MUC16×CD3 bispecifics. All of the testedbispecific antibodies activated and directed human T cells to depletethe target cells with EC₅₀s in picomolar range. Additionally, theobserved target-cell lysis (depletion) was associated with anup-regulation of CD69 (or CD25) on CD2+ T cells, also with picomolar(pM) EC₅₀s.

Importantly, the results of this example also demonstrate that abispecific antibody constructed with a CD3 binding arm that displayedweak-to-no measurable binding to CD3 protein or CD3-expressing cells(i.e. CD3-VH-G5) still retained the ability to activate T-cells andexhibited potent cytotoxicity of tumor antigen-expressing cells.

TABLE 8A Cytotoxicity and T-cell activation properties of selected MUC16× CD3 Bispecific Antibodies Bispecific OVCAR3 T cell activation AntibodyAnti-CD3 cell depletion (CD69 upregulation) Identifier Binding Arm EC₅₀[M] EC₅₀ [M] BSMUC16/ CD3-VH-G 2.24E−11 5.88E−12 CD3-001 BSMUC16/CD3-VH-G5 3.06E−11 1.01E−11 CD3-002

TABLE 8B Cytotoxicity and T-cell activation properties of selected MUC16× CD3 Bispecific Antibodies Bispecific PEO-1 cell T cells activation Tcells activation Antibody depletion (CD69 upregulation) (CD25upregulation) Identifier EC50 [M] EC50 [M] EC50 [M] BSMUC16/ 2.56E−118.34E−12 3.90E−11 CD3-001 BSMUC16/ 6.75E−11 1.34E−11 8.89E−11 CD3-002BSMUC16/ 7.74E−11 1.72E−11 1.06E−10 CD3-005

TABLE 8C Cytotoxicity and T-cell activation properties of selected MUC16× CD3 Bispecific Antibodies Bispecific OVCAR3-Luc T cells activation Tcells activation Antibody cell depletion (CD69 upregulation) (CD25upregulation) Identifier EC50 [M] EC50 [M] EC50 [M] BSMUC16/ 1.54E−112.98E−12 3.06E−11 CD3-001 BSMUC16/ 5.16E−11 1.54E−11 1.17E−10 CD3-005

Example 6: Hydrogen/Deuterium (H/D) Exchange Based Epitope Mapping ofAnti-Muc16 Antibodies H4sH8767P, H1H8794P2, and H1H8799P2 Binding to aPortion of the C-Term Domain of hMUC16

Experiments were conducted to determine the MUC16 amino acid residueswithin the C-terminal five SEA domains (SEQ ID No: 1902, hereafterreferred to as hMUC16.nub), with which the anti-MUC16 antibodiesH4sH8767P, H1H8794P2, and H1H8799P2 interact. For this purpose,Hydrogen/Deuterium (H/D) Exchange epitope mapping with mass spectrometry(HDX-MS) was utilized. A general description of the H/D exchange methodis set forth in Ehring (1999) Analytical Biochemistry 267(2):252-259;and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

HDX-MS experiments were performed on an integrated Waters HDX/MSplatform, consisting of a Leaptec HDX PAL system for deuterium labeling,a Waters Acquity M-Class (Auxiliary solvent manager) for sampledigestion and loading, a Waters Acquity M-Class (pBinary solventmanager) for the analytical column gradient, and a Synapt G2-Si massspectrometer for peptic peptide mass measurement.

The labeling solution was prepared in 10 mM PBS buffer in D₂O at pD 7.0.For deuterium labeling, 3.8 μL of hMUC16.nub (12 pmol/μL) or hMUC16.nubpremixed with the anti-MUC16 antibody H4sH8767P, H1H8794P2, or H1H8799P2in a 2:1 molar ratio was incubated with 56.2 μL. D₂O labeling solutionfor various time-points (e.g.: undeuterated control=0 sec; deuteriumlabeling: 1 min and 20 min). The deuteration was quenched bytransferring 50 μL sample to 50 μL pre-chilled quench buffer (0.2 MTCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and themixed sample was incubated at 1.0° C. for two minutes. The quenchedsample was then injected into a Waters HDX Manager for onlinepepsin/protease XIII digestion. The digested peptides were trapped ontoan ACQUITY UPLC BEH C18 1.7-μm, 2.1×5 mm VanGuard pre-column at 0° C.and eluted to an analytical column ACQUITY UPLC BEH C18 (1.7-μm, 1.0×50mm) for a 9-minute gradient separation of 5%-40% B (mobile phase A: 0.1%formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile).The mass spectrometer used a cone voltage of 37V, a scan time of 0.5 s,and mass/charge range of 50-1700 Th.

For the identification of the peptide residues of hMUC16.nub with whichH4sH8767P, H1H8794P2, and H1H8799P2 interact, LC-MSE data from theundeuterated sample were processed and searched against a database thatincluded sequences for hMUC16.nub, pepsin and a randomized sequenceusing Waters ProteinLynx Global Server (PLGS) software. The identifiedpeptides were imported to DynamX software and filtered by twocriteria: 1) minimum products per amino acid: 0.25 and 2) replicationfile threshold: 2. DynamX software then automatically determineddeuterium uptake of each peptide based on retention time and high massaccuracy (<10 ppm) across multiple time points with 3 replicates at eachtime.

Using the online pepsin/protease XIII column coupled with MSE dataacquisition, a total of 109 peptides from hMUC16.nub were identified inthe absence or presence of the H4sH8767P, representing 64% sequencecoverage. Six peptides had significantly reduced deuteration uptake(centroid delta values >0.5 daltons from at least one time point withp-values <0.05) when bound to H4sH8767P and are illustrated in the Table9A. The recorded peptide mass corresponds to the average value of thecentroid MH⁺ mass from three replicates. These peptides, correspondingto amino acids 428-434, 453-467, and 474-481 of hMuc16.nub, had slowerdeuteration rates when bound to H4sH8767P. These identified residuesalso correspond to residues 14237-14243, 14262-14276, and 14283-14290 ofhMUC16 as defined by the Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ IDNO:1899.

TABLE 9AhMUC16.nub Peptides with Altered Deuteration Rates Upon Binding H4sH8767P1 min Deuteration 20 min Deuteration Residues of Amino hMUC16.nubhMUC16.nub SEQ ID Acid + + NO: 1902 Sequence hMUC16.nub H4sH8767P ΔhMUC16.nub H4sH8767P Δ 428-434 LYKGSQL 809.97 ± 809.07 ± −0.26 811.05 ±810.17 ± −0.88 0.03 0.06 0.16 0.01 429-434 YKGSQL 697.10 ± 696.74 ±−0.35 698.13 ± 697.60 ± −0.52 0.00 0.00 0.02 0.03 453-467 VTVKALFS1595.35 ± 1593.97 ± −1.38 1596.01 ± 1595.33 ± −0.68 SNLDPSL 0.08 0.20.08 0.03 459-467 FSSNLDPS 983.19 ± 981.57 ± −1.62 983.51 ± 982.76 ±−0.75 L 0.01 0.08 0.03 0.07 460-467 SSNLDPSL 835.44 ± 834.01 ± −1.43835.89 ± 835.12 ± −0.74 0.01 0.00 0.01 0.15 474-481 DKTLNASF 899.76 ±899.25 ± −0.51 900.63 ± 900.10 ± −0.54 0.00 0.06 0.00 0.03

Using the online pepsin/protease XIII column coupled with MS^(E) dataacquisition, a total of 109 peptides from hMUC16.nub were identified inthe absence or presence of the H1H8794P2, representing 64% sequencecoverage. Three peptides had significantly reduced deuteration uptake(centroid delta values >0.5 daltons from at least one time point withp-values <0.05) when bound to H1H8794P2 and are illustrated in the Table9B. The recorded peptide mass corresponds to the average value of thecentroid MH⁺ mass from three replicates. These peptides, correspondingto amino acids 126-131, 127-131, and 132-138 of hMuc16.nub, had slowerdeuteration rates when bound to H1H8794P2. These identified residuesalso correspond to residues 13935-13940, 13936-13940, and 13941-13947 ofhMUC16 as defined by the Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ IDNO:1899.

TABLE 9B hMUC16.nub Peptides with Altered DeuterationRates Upon Binding H1H8794P2 1 min Deuteration 20 min DeuterationResidues of Amino  hMUC16.nub hMUC16.nub SEQ ID Acid  + + NO: 1902Sequence hMUC16.nub H1H8794P2 Δ hMUC16.nub H1H8794P2 Δ 126-131 LRYMAD771.41 ± 770.60 ± −0.81 771.91 ± 770.76 ± −1.15   0.01 0.04 0.04 0.02127-131 RYMAD 658.03 ± 657.32 ± −0.71 657.89 ± 657.27 ± −0.6   0.02 0.010.01 0.01 132-138 MGQPGSL 692.55 ± 691.42 ± −1.13 692.61 ± 691.58 ±−1.03   0.02 0.15 0.01 0.02

Using the online pepsin/protease XIII column coupled with MSE dataacquisition, a total of 109 peptides from hMUC16.nub were identified inthe absence or presence of the H1H8799P2, representing 64% sequencecoverage. Four peptides had significantly reduced deuteration uptake(centroid delta values >0.5 daltons from at least one time point withp-values <0.05) when bound to H1H8799P2 and are illustrated in the Table9C. The recorded peptide mass corresponds to the average value of thecentroid MH⁺ mass from three replicates. These peptides, correspondingto amino acids 357-369, 358-366, 358-369, and 361-369 of hMuc16.nub, hadslower deuteration rates when bound to H1H8799P2. These identifiedresidues also correspond to residues 14165-14178, 14166-14176,14166-14178, and 14170-14178 of hMUC16 as defined by the Uniprot entryQ8WXI7 (MUC16_HUMAN), SEQ ID NO:1899.

TABLE 9C hMUC16.nub Peptides with Altered DeuterationRates Upon Binding H1H8799P2 1 min Deuteration 20 min DeuterationResidues of Amino hMUC16.nub hMUC16.nub SEQ ID Acid + + NO: 1902Sequence hMUC16.nub H1H8799P2 Δ hMUC16.nub H1H8799P2 Δ 357-369 LSQLTHGVT1404.15 ± 1403.41 ± −0.74 1406.14 ± 1404.26 ± −2.11 QLGF 0.03 0.09 0.150.02 358-366 SQLTHGVT 972.37 ± 972.04 ± −0.33 973.94 ± 972.56 ± −1.38 QL0.10 0.10 0.03 0.00 358-369 SQLTHGVT 1291.23 ± 1290.20 ± −1.03 1291.34 ±1291.05 ± −2.27 QLGF 0.02 0.00 0.02 0.06 361-369 THGVTQLG 1404.15 ±1403.42 ± −0.73 1406.14 ± 1404.03 ± −2.11 F 0.03 0.05 0.14 0.02

Example 7: Pharmacokinetic Assessment of Anti-MUC16×CD3 BispecificAntibodies

Assessment of the pharmacokinetics of anti-MUC16×CD3 bispecificantibodies BSMUC16/CD3-001 and BSMUC16/CD3-005 and an isotype controlwere conducted in humanized MUC16×CD3 mice (mice homozygous for humanMUC16 and CD3 expression, MUC16^(hu/hu)×CD3^(hu/hu)), CD3 humanized mice(mice homozygous for human CD3 expression, CD3^(hu/hu)) andstrain-matched (75% C57BL, 25%129Sv) wild-type (WT) mice. Cohortscontained 4-5 mice per tested antibody and per mouse strain. All micereceived a single intra-peritoneal (i.p.) 0.4 mg/kg dose. Blood sampleswere collected at 3 and 6 hours, 1, 3, 7, 14 and 28 days post dosing.Blood was processed into serum and frozen at −80° C. until analyzed.

Circulating antibody concentrations were determined by total human IgGantibody analysis using the GyroLab xPlore™ (Gyros, Uppsala, Sweden).Briefly, a biotinylated goat anti-human IgG polyclonal antibody (JacksonImmunoResearch, West Grove, Pa.) was captured onto streptavidin coatedbeads on a Gyrolab Bioaffy 200 CD (Gyros) in order to capture the humanIgG present in the sera. After affinity column capture, bound human IgGantibody in samples was detected with Alexa-647 labeled goat anti-humanIgG (Jackson ImmunoResearch). Fluorescent signal on the column allowedfor the detection of bound IgG and response units (RU) were read by theinstrument. Sample concentrations were determined by interpolation froma standard curve that was fit using a 5-parameter logistic curve fitusing the Gyrolab Evaluator Software. PK parameters were determined bynon-compartmental analysis (NCA) using Phoenix®WinNonlin® softwareVersion 6.3 (Certara, L.P., Princeton, N.J.) and an extravascular dosingmodel. Using the respective mean concentration values for each antibody,all PK parameters including observed maximum concentration in serum(C_(max)), estimated half-life observed (t½), and area under theconcentration curve versus time up to the last measureable concentration(AUC_(last)) were determined using a linear trapezoidal rule with linearinterpolation and uniform weighting

Following i.p. administration of antibodies in WT mice, the total IgGconcentration-time profiles of BSMUC16/CD3-001, BSMUC16/CD3-005 and theisotype control were all similar, characterized first by a brief drugdistribution followed by a single drug elimination phase throughout theremainder of the study. Maximum serum concentrations (C_(max)) andcalculated drug exposure (AUC_(last)) of the three antibodies werecomparable (within 1.3-fold of each other).

Following i.p. administration of antibodies in CD3^(hu/hu) mice,BSMUC16/CD3-001, BSMUC16/CD3-005 and isotype control had comparableC_(max) concentrations (4.6, 3.6 and 4.1 μg/mL, respectively).BSMUC16/CD3-005 and the isotype control exhibited similar drugelimination curves, while BSMUC16/CD3-001 exhibited steeper drugelimination than both, suggesting that human CD3 target binding drivesclearance. Terminal antibody concentration for BSMUC16/CD3-001 was 0.03μg/mL, which is about 28-fold less than terminal antibody concentrationsdetermined for the isotype control (0.85 μg/mL) and 22-fold less thanBSMUC16/CD3-005 (0.66 μg/mL) serum concentrations.

In MUC16^(hu/hu)×CD3^(hu/hu) double-humanized mice, the Muc16×CD3bispecific and isotype control antibodies had comparable C_(max)concentrations (C_(max) range: 4.5-6.9 μg/mL). Both bispecificantibodies exhibited steeper drug elimination than the isotype controlsuggesting a target-mediated effect. Terminal antibody concentrationsfor BSMUC16/CD3-001 and BSMUC16/CD3-005 were about 29-fold and 2.9-foldless, respectively, than terminal antibody concentrations determined forthe isotype control (0.86 μg/mL).

A summary of the data for total anti-MUC16×CD3 bispecific antibodies andisotype control antibody concentrations are summarized in Table 10. MeanPK parameters are described in Tables 11A and 11B. Mean total antibodyconcentrations versus time are shown in FIGS. 1, 2 , and 3. Inconclusion, MUC16×CD3 bispecific antibodies exhibited similar C_(max)and drug elimination curves in WT mice, but BSMUC16/CD3-001 displayedsteeper elimination rates than BSMUC16/CD3-005 and the isotype controlin CD3 single-humanized mice and MUC16/CD3 double humanized mice. Sincethe bispecific antibodies administered in this PK study are comprised ofthe same anti-MUC16 binding arm, the results suggest that the strengthof binding of the CD3 targeting arm may play a role in drug exposurelevels (AUC_(last)) and drug elimination rates. Neither BSMUC16/CD3-001or BSMUC16/CD3-005 bind mouse MUC16 or mouse CD3.

TABLE 10 Mean Concentrations of Total IgG in Serum Following a Single0.4 mg/kg Intraperitoneal Injection of BSMUC16/CD3-001, BSMUC16/CD3-005and Isotype Control Antibodies in WT Mice, Humanized CD3 Mice andHumanized MUC16 × CD3 mice Total mAb Concentration In Mouse Serum WTCD3^(hu/hu) MUC16^(hu/hu) × CD3^(hu/hu) Time Mean Mean Mean Antibody (d)(μg/mL) +/−SD (μg/mL) +/−SD (μg/mL) +/−SD BSMUC16/ 0.13 5.39 0.34 4.300.29 6.77 1.52 CD3-001 0.25 5.80 0.36 4.26 1.07 6.63 1.06 1.00 4.13 0.432.87 0.71 4.89 0.53 3.00 3.19 0.53 1.44 0.27 2.50 0.22 7.00 2.61 0.730.72 0.13 1.20 0.22 14.00 1.44 0.69 0.18 0.05 0.28 0.08 21.00 0.93 ND0.07 0.02 0.06 0.05 28.00 0.60 ND 0.04 0.01 0.03 0.02 BSMUC16/ 0.13 4.230.62 3.35 1.15 4.35 0.24 CD3-005 0.25 4.53 0.55 3.40 0.96 4.45 0.49 1.003.47 0.32 2.72 0.42 3.00 0.61 3.00 2.51 0.13 1.95 0.37 1.98 0.41 7.002.02 0.24 2.31 0.67 1.58 0.36 14.00 1.19 0.17 1.01 0.23 0.78 0.26 21.001.19 0.29 1.19 0.11 0.66 0.29 28.00 0.71 0.20 0.66 0.28 0.30 0.22Isotype 0.13 5.07 1.16 5.43 1.30 6.56 0.70 Control 0.25 5.91 1.10 5.671.91 6.48 0.90 1.00 2.64 0.24 2.98 1.14 2.82 0.30 3.00 2.05 0.06 2.290.83 1.57 0.37 7.00 1.80 0.25 2.14 0.85 1.96 0.37 14.00 1.22 0.28 1.480.66 1.34 0.37 21.00 1.20 0.58 1.43 0.72 1.24 0.44 28.00 0.73 0.24 0.850.29 0.86 0.41 Time: (h, when noted) = time in hours post single-doseinjection; D = Day of study; SD = Standard deviation; ND = Notdetermined due to exclusion of mice with drug clearing anti-drug titers

TABLE 11A Summary of Pharmacokinetic Parameters: CD3^(hu/hu) humanizedmice WT mice CD3^(hu/hu) mice Param- Isotype BSMUC16/ BSMUC16/ IsotypeBSMUC16/ BSMUC16/ eter Units Control CD3-001 CD3-005 Control CD3-001CD3-005 C_(max) μg/mL 5 ± 3  6 ± 0.4   5 ± 0.5 4.1 ± 3  4.6 ± 0.8 3.5 ±1  T_(1/2) d 11 ± 4  7 ± 3 12 ± 2  14 ± 0.5 3.9 ± 0.6 11 ± 5 AUC_(last)d · μg/mL 35 ± 18 40 ± 11 45 ± 5 49 ± 20 16 ± 3   36 ± 13 C_(max) = Peakconcentration; AUC = Area under the concentration-time curve; AUC_(last)= AUC computed from time zero to the time of the last positiveconcentration; T_(1/2) = Estimated half-life observed

TABLE 11B Summary of Pharmacokinetic Parameters: MUC16^(hu/hu) ×CD3^(hu/hu) double-humanized mice WT mice MUC16^(hu/hu) × CD3^(hu/hu)mice Param- Isotype BSMUC16/ BSMUC16/ Isotype BSMUC16/ BSMUC16/ eterUnits Control CD3-001 CD3-005 Control CD3-001 CD3-005 C_(max) μg/mL 5 ±3  6 ± 0.4   5 ± 0.5 6.7 ± 0.7 6.9 ± 1  4.5 ± 4 T_(1/2) d 11 ± 4  7 ± 312 ± 2 12.9 ± 4   3.3 ± 0.8 8.2 ± 4 AUC_(last) d · μg/mL 35 ± 18 40 ± 1145 ± 5 46 ± 10 27 ± 3   34 ± 11 C_(max) = Peak concentration; AUC = Areaunder the concentration-time curve; AUC_(last) = AUC computed from timezero to the time of the last positive concentration; T_(1/2) = Estimatedhalf-life observed

Example 8: Anti-MUC16/Anti-CD3 Bispecific Antibodies Display PotentAnti-Tumor Efficacy In Vivo

To determine the in vivo efficacy of exemplary anti-MUC16/anti-CD3bispecific antibodies identified as having weak or no detectable bindingto human and cynomolgus CD3, studies were performed in immunocompromisedmice bearing human prostate cancer xenografts. The efficacy of selectedbispecific antibodies was tested in both immediate treatment andtherapeutic treatment dosing models.

Efficacy of Anti-MUC16/Anti-CD3 Bispecific Antibodies in Human TumorXenograft Models

To assess the in vivo efficacy of the anti-MUC16/anti-CD3 bispecifics inhuman tumor xenograft studies, NOD scid gamma (NSG) mice (JacksonLaboratories, Bar Harbor, Me.) were pre-implanted with human peripheralblood mononuclear cells (PBMCs; ReachBio LLC., Seattle, Wash.) and thengiven ascites cells from the human ovarian cancer cell line OVCAR-3(American Type Tissue Culture, Manassas, Va.) transduced with luciferase(OVCAR-3/Luc). OVCAR-3 cells endogenously express MUC-16.

Briefly, NSG mice were injected intraperitoneally (i.p.) with 5.0×10⁶human PBMCs. 8 d later, 1.5×10⁶ ascites cells from the OVCAR-3/Luc cellline, previously passaged in vivo, were administered i.p. to the NSGmice engrafted with PBMCs. In the immediate treatment group, mice weretreated i.p. on the day of OVCAR-3/Luc cell implantation with MUC16/CD3Bispecific antibodies BSMUC16/CD3-001 or BSMUC16/CD3-005, or an isotypecontrol, at a dose of 10 μg/mouse (N=5 mice/treatment group). In thetherapeutic dose model, mice were treated i.p. 7 d post tumorimplantation with the MUC16/CD3 Bispecific or control antibodiesdescribed above, at a dose of 10 μg/mouse (N=5/treatment group).

In all studies, tumor growth was monitored via bioluminescent imaging(BLI). Mice were injected i.p. with the luciferase substrate D-luciferinsuspended in PBS (150 mg/kg) and imaged under isoflurane anesthesiaafter 10 min. BLI was performed using the Xenogen IVIS system (PerkinElmer, Hopkinton, Mass.) and BLI signals were extracted using LivingImage software (Xenogen/Perkin Elmer). Regions of interest were drawnaround each cell mass and photon intensities were recorded as photons(p)/sec (s)/cm²/steradian (sr). For the immediate-treatment group, datais shown as BLI levels 26 d post tumor implantation (Table 12A). For thetherapeutic-treatment group, data is shown as fold-change in BLI betweenday 6 (1 d before treatment) and at study endpoint (26 d post tumorimplantation; Table 12B).

As the results show, both BSMUC16/CD3-001 and BSMUC16/CD3-005 showedsimilar efficacy in suppressing tumor growth compared to the isotypecontrol when BLI was measured at Day 26 in the immediate dosing model.Both anti-MUC16/anti-CD3 bispecific antibodies also suppressed thegrowth of established tumors when administered 7 d post tumorimplantation, compared to the control. In summary, the bispecificanti-MUC16/anti-CD3 antibodies of this invention display potentanti-tumor efficacy in several models.

TABLE 12A Efficacy of anti-MUC16/anti-CD3 Bispecific Antibodies inImmune-Compromised Xenograft Model: Immediate Dosing Avg BioluminescentRadiance Tumor Model/ Bispecific (photons/sec/ Mouse Strain/ Antibodycm²/steradian) Dose Identifier N Day 26 (mean ± SD) OVCAR-3/Luc/BSMUC16/CD3-001 5 1.4 × 10³ ± 3.5 × 10² NSG/ BSMUC16/CD3-005 5 1.5 × 10³± 9.7 × 10² 10 μg/mouse Isotype Control 5 2.0 × 10⁷ ± 1.0 × 10⁶

TABLE 12B Efficacy of anti-Muc16/anti-CD3 Bispecific Antibodies inImmune-Compromised Xenograft Model: Therapeutic Treatment Fold change inAvg Bioluminescent Radiance Tumor Model/ Bispecific [p/s/cm²/sr] MouseStrain/ Antibody at Day 26 relative to Dose Identifier N Day 6 (mean ±SD) OVCAR-3/Luc/ BSMUC16/CD3-001 5 2.0 ± 5.0 NSG/ BSMUC16/CD3-005 5 0.01± 0.02 10 μg/mouse Isotype Control 5 21.0 ± 8.0 

In further experiments, the in vivo efficacy of an anti-MUC16/anti-CD3bispecific antibody was evaluated in xenogenic and syngeneic tumormodels. For the first xenogenic model, NSG mice were injectedintraperitoneally (IP) with OVCAR-3/Luc cells previously passaged invivo (Day 0) eleven days after engraftment with human PBMCs. Mice weretreated IP with 0.01, 0.1, or 0.5 mg/kg BSMUC16/CD3-001, or administered0.5 mg/kg non-binding control or CD3-binding control on Days 6, 10, 13,16, and 21. Tumor burden was assessed by BLI on Days 6, 14, and 20 posttumor implantation. Treatment with 0.1 or 0.5 mg/kg of BSMUC16/CD3-001resulted in significant anti-tumor efficacy as determined by BLImeasurements on Day 20, as shown in Tables 13A-C and in FIGS. 4-6 . Forthe second xenogenic model, NSG mice were injected with OVCAR-3/Luccells previously passaged in vivo (Day 0) thirteen days afterengraftment with human PBMCs and a second batch of PBMCs weretransferred on Day 4. Mice were treated intravenously (IV) with 0.1,0.5, 1, or 5 mg/kg BSMUC16/CD3-005 or administered 5 mg/kg non-bindingcontrol or CD3-binding control on Days 5, 8, 12, 15, 19 and 22. Tumorburden was assessed by BLI on Days 4, 11, 18 and 25. Treatment with 0.5,1, or 5 mg/kg BSMUC16/CD3-005 resulted in significant anti-tumorefficacy as shown by BLI measurements and fold changes (Tables 13D-F andFIGS. 7-9 ). To examine efficacy in an immune-competent model, themurine CD3 gene was replaced with human CD3 and a portion of the mouseMUC16 gene was replaced with the human sequence. The replacementsresulted in a mouse whose T cells express human CD3 and that expresses achimeric MUC16 molecule containing a portion of human MUC16 where theBSMUC16/CD3-001 bispecific antibody binds. For the syngeneic tumormodel, ID8-VEGF cell lines engineered to express the portion of humanMUC16 were used. Mice were implanted with the ID8-VEGF/huMUC16 cellssubcutaneously and treated with 100 μg of BSMUC16/CD3-001 either on dayof implantation or ten days after implantation when tumors wereestablished. Treatment with 100 μg BSMUC16/CD3-001 resulted insignificant anti-tumor efficacy, as shown in Table 13G and FIG. 10 .

Implantation and measurement of xenograft tumors: Ascites cells from theOVCAR-3/Luc cell line, previously passaged in vivo, were administered IPinto the NSG mice previously engrafted with human PBMCs. BLI wasmeasured as a read-out for tumor growth several days after OVCAR-3/Lucimplantation and at multiple times during study. After initial BLImeasurement for cohorting, mice were divided into groups of 4-6 animalseach and administered MUC16×CD3 bispecific or control antibodies twiceper week throughout the study.

Calculation of xenograft tumor growth and inhibition: Bioluminescenceimaging was used to measure tumor burden. Mice were injected IP with 150mg/kg (as determined by body weights at the start of the experiment) ofthe luciferase substrate D-luciferin suspended in PBS. Ten minutes afterdosing, BLI imaging of the mice was performed under isofluraneanesthesia using the Xenogen IVIS system. Image acquisition was carriedout with the field of view at D, subject height of 1.5 cm, and mediumbinning level for 0.5-min exposure time. BLI signals were extractedusing Living Image software. Regions of interest were drawn around eachtumor mass and photon intensities were recorded as p/s/cm²/sr.Statistical analysis was performed using GraphPad Prism software(Version 6). Statistical significance for the BLI results was determinedusing an unpaired nonparametric Mann-Whitney t-test. Fold changes werecalculated by the formula: (Day20-Day6)/Day6 for study 1 and(Day25−Day4)/Day4 for study 2.

Implantation and measurement of syngeneic tumors: Mice expressing humanCD3 and a human-murine chimera of MUC16 in the corresponding mouse lociwere implanted with 10e6 ID8-VEGF/huMUC16 cells subcutaneously (SC).Mice were administered BSMUC16/CD3-001 or a CD3-binding control IP,twice per week throughout study. Treatment began on Day 0 or Day 10 postimplantation. Tumor growth was measured with calipers twice per week.Mice were sacrificed 47 days after tumor implantation.

Calculation of syngeneic tumor growth and inhibition: In order todetermine tumor volume by external caliper, the greatest longitudinaldiameter (length) and the greatest transverse diameter (width) weredetermined. Tumor volume based on caliper measurements were calculatedby the formula: Volume=(length×width²)/2. Statistical significance wasdetermined using an unpaired nonparametric Mann-Whitney t-test.

The anti-tumor efficacy of the BSMUC16/CD3-001 bispecific antibody inthe xenogenic and syngeneic in vivo tumor models is shown in Tables13A-D, below.

TABLE 13A OVCAR-3 model study 1. Level of Bioluminescence on Day 6 posttumor implantation Avg Radiance [p/s/cm2²/sr] 6 days post-implantationAntibody (mg/kg) (median ± SEM) non-binding control (0.5) 8.15e+05 ±7.88e+04 CD3-binding control (0.5) 6.39e+05 ± 8.67e+04 BSMUC16/CD3-001(0.5) 7.64e+05 ± 1.19e+05 BSMUC16/CD3-001 (0.1) 6.31e+05 ± 1.10e+05BSMUC16/CD3-001 (0.01) 8.77e+05 ± 7.91e+04

TABLE 13B OVCAR-3 model study 1. Level of Bioluminescence on Day 20 posttumor implantation Avg Radiance [p/s/cm2²/sr] 20 days post-implantationAntibody (mg/kg) (median ± SEM) non-binding control (0.5) 8.63e+06 ±1.45e+06 CD3-binding control (0.5) 9.94e+06 ± 1.08e+06 BSMUC16/CD3-001(0.5) 9.37e+02 ± 9.62e+02 BSMUC16/CD3-001 (0.1) 2.36e+04 ± 1.28e+06BSMUC16/CD3-001 (0.01) 6.51e+06 ± 1.60e+06

TABLE 13C OVCAR-3 model study 1. Fold Change in BLI between Day 6 andDay 20 post tumor implantation Fold change in Avg Radiance [p/s/cm2²/sr]from Day 6 to D 20 post-implantation Antibody (mg/kg) (mean ± SD)non-binding control (0.5) 9.5 ± 1.9 CD3-binding control (0.5) 15.6 ±6.7  BSMUC16/CD3-001 (0.5) −1.00 ± 0.00  BSMUC16/CD3-001 (0.1) 1.2 ± 4.7BSMUC16/CD3-001 (0.01) 5.6 ± 4.2

TABLE 13D OVCAR-3 model study 2. Level of Bioluminescence on Day 4 posttumor implantation Avg Radiance [p/s/cm2²/sr] 4 days post-implantationAntibody (mg/kg) (median ± SEM) non-binding control (5) 1.54e+05 ±9.93e+03 CD3-binding control (5) 1.34e+05 ± 1.55e+04 BSMUC16/CD3-005 (5)1.54e+05 ± 1.03e+04 BSMUC16/CD3-005 (1) 1.38e+05 ± 4.65e+03BSMUC16/CD3-005 (0.5) 1.31e+05 ± 4.03e+03 BSMUC16/CD3-005 (0.1) 1.53e+05± 1.93e+04

TABLE 13E OVCAR-3 model study 2. Level of Bioluminescence on Day 25 posttumor implantation Avg Radiance [p/s/cm2²/sr] 25 days Antibody (mg/kg)post-implantation (median ± SEM) non-binding control (5) 7.20e+06 ±8.91e+05 CD3-binding control (5) 6.15e+06 ± 7.26e+05 BSMUC16/CD3-005 (5)1.52e+03 ± 4.86e+05 BSMUC16/CD3-005 (1) 6.99e+03 ± 6.23e+03BSMUC16/CD3-005 (0.5) 2.23e+03 ± 2.35e+05 BSMUC16/CD3-005 (0.1) 7.63e+06± 1.83e+06

TABLE 13F OVCAR-3 model study 2. Fold Change in BLI between Day 4 andDay 25 post tumor implantation Fold change in Avg Radiance [p/s/cm2²/sr]from Day 4 to D25 Antibody (mg/kg) post-implantation (mean ± SD)non-binding control (5) 46.8 ± 20.6 CD3-binding control (5) 55.0 ± 14.7BSMUC16/CD3-005 (5) 2.5 ± 8.5 BSMUC16/CD3-005 (1) −0.9 ± 0.1 BSMUC16/CD3-005 (0.5) 0.7 ± 3.6 BSMUC16/CD3-005 (0.1) 45.4 ± 35.7

TABLE 13G ID8-VEGF/huMUC16 model. Tumor size (mm3) at Day 47 Tumor size(mm3) at Treatment start Antibody (μg) Day 47 (mean ± SEM) Day 0CD3-binding control (100) 827.5 ± 223.5 Day 0 BSMUC16/CD3-001 (100) 51.2± 51.2 Day 10 BSMUC16/CD3-001 (100) 273.8 ± 92.36

Example 9: Conjugate Preparation and Characterization

All the monoclonal antibodies were expressed in CHO cells and purifiedby Protein A. An isotype control was also prepared in a similar fashion.The non-binding isotype control antibody was derived from animmunological antigen having no relation to oncology.

The antibody (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was treatedwith 1 mM dithiothreitol at 37° C. for 30 min. After gel filtration(G-25, pH 4.5 sodium acetate), one of the maleimido linker payloadderivatives Compound 7 or Compound 10 (see Table 14) (1.2 equivalents/SHgroup) in DMSO (10 mg/ml) was added to the reduced antibody and themixture adjusted to pH 7.0 with 1 M HEPES (pH 7.4). Compound 7 andCompound 10, and methods of making the compounds, are described in PCTPublication No. WO2014/145090, published on Sep. 18, 2014 and PCTPublication No. WO2016/160615, published on Oct. 6, 2016, respectively,each of which is entirely incorporated herein by reference. After 1 hthe reaction was quenched with excess N-ethyl maleimide. The conjugateswere purified by size exclusion chromatography and sterile filtered.Protein and linker payload concentrations were determined by UV spectralanalysis. Size-exclusion HPLC established that all conjugates usedwere >95% monomeric. Yields are reported in Table 14 based on proteindeterminations. All conjugated antibodies were analyzed by UV for linkerpayload loading values according to Hamblett et al, Cancer Res 2004 107063. The results are summarized in Table 14.

A conjugate comprising Compound 60 can be prepared using a similarmethod. Compound 60, and methods of making the compound, is described inPCT Publication No. WO2016/160615 (Example 20), published on Oct. 6,2016, which is entirely incorporated herein by reference. Compound 60 isMaytansin-N-methyl-L-alanine-(3-methoxy-4-amino)benzamido-Cit-Val-Cap-Mal.

TABLE 14 Summary of Payload (Chemotoxic Drug) andAntibody-Drug-Conjugate Parameters Compound ε252 nm (cm⁻¹ M⁻¹) ε280 nm(cm⁻¹ M⁻¹) 7 [Maytansin-3-N-methyl-L- 50600 8100(S)-alanine-propanamidyl-3- N-methyl-N-[4- (amino-citrulline-valine-hexanamide-6- maleimidy/)benzyl]carbamate] 10 [Maytansin-N-methyl-L-45990 20600 alanine-4-aminobenzamide- citrulline-valine-caprolyl-6-maleimidyl] Antibody ε252 nm (cm⁻¹ M⁻¹) ε280 nm (cm⁻¹ M⁻¹) H1H9519N83995 235280 H1H9521N 85564 232050 Isotype Control 75113 218360Payload:Antibody Antibody Conjugate (UV) Yield % H1H9519N-7 3.5 40H1H9521N-7 3.6 40 H1H9521N-10 3.0 40 Isotype Control-7 3.0 60 IsotypeControl-10 3.4 60

Example 10: Anti-MUC16 Antibody Drug Conjugates are Potent Inhibitors ofTumor Growth in In Vivo MUC16-Positive Prostate Cancer Xenograft Models

To determine the in vivo efficacy of the anti-MUC16 antibodiesconjugated to Compound 7 and Compound 10, studies were performed inimmunocompromised mice bearing MUC16 positive ovarian cancer xenografts.

For these studies, female SCID mice (Taconic, Hudson N.Y.) wereimplanted with OVCAR3 [NIH:OVCAR-3 (OVCAR3, ATCC HTB-161)] cellstransfected with luciferase (OVCAR3/luc) that endogenously expressMUC16. For intraperitoneal (IP) tumors, mice were randomized intotreatment groups, and dosed with either anti-MUC16 drug conjugatedantibodies (see Example 9), a non-binding conjugated antibody, orvehicle following detection of tumor luminescent signal. Forsubcutaneous (SC) xenografts, once tumors had reached an average volumeof 200 mm³ (˜Day 16), mice were randomized into treatment groups, anddosed with either anti-MUC16 drug conjugated antibodies, a non-bindingconjugated antibody, or vehicle. In these in vivo studies, antibodieswere dosed and tumors were then monitored until ascites developed or anaverage tumor size of approximately 1200 mm³ was attained in the cohortdosed with vehicle alone. At this point the Tumor Growth Inhibition wascalculated.

In an initial intraperitoneal (IP) study, exemplary anti-MUC16antibodies conjugated to Compound 7 were examined for efficacy inreducing OVCAR3/luc luminescent signal. Mice received four once weeklydoses of anti-MUC16 and control ADCs at 85 μg/kg of drug equivalentbased on ADC drug:antibody ratios. As summarized in Table 15A,H1H9519N-Compound 7 and H1H9521N-Compound 7 potently inhibited ascitestumor growth. These anti-MUC16 ADCs efficiently reduced tumor size, witha 100 percent reduction in tumor luminescence relative to vehiclecontrol. The Control ADC did not mediate any inhibition of OVCAR/lucascites tumor cell growth.

A further study assessing the efficacy of anti-MUC16 ADCs againstsubcutaneous (SC) OVCAR3/luc tumor is summarized in Table 15B. Micereceived four once weekly doses of anti-MUC16 and control ADCs at 85μg/kg of drug equivalent based on ADC drug:antibody ratios. Whenconjugated to Compound 7, MUC16 ADCs H1H9519N-Compound 7 andH1H9521N-Compound 7 again produced significant anti-tumor effect; thistime against the subcutaneous OVCAR3/luc tumors. Accordingly, these ADCsmediated a 100% and 109% inhibition of tumor growth respectively.Control ADC did not mediate any inhibition of OVCAR/luc subcutaneoustumor growth.

In a third study, the efficacy of the anti-MUC16 antibody H1H9521Nconjugated to the linker drug Compound 10 was assessed in the IPOVCAR3/luc tumor model. Mice received single doses of anti-MUC16 andcontrol ADCs at 85 μg/kg, 170 μg/kg and 340 μg/kg of drug equivalentbased on ADC drug:antibody ratios. As summarized in Table 15C,H1H9519N-10 potently inhibited ascites tumor growth. The doses ofH1H9519N-Compound 10 resulted in a 99-100% inhibition of tumorluminescence relative to vehicle control. Some inhibition was observedwith Control ADC using Compound 10 although this was more moderate thanthat observed following anti-MUC16 H1H9519N-Compound 10.

TABLE 15A Inhibition of OVCAR3/luc IP Tumor Growth at Day 49 in SCIDmice treated with anti-MUC16 antibodies conjugated to Compound 7 FinalTumor Average Tumor Average Radiance Growth Inhibition Treatment Group(mean ± SEM) (%) Vehicle 16469750 ± 10679335 0 Control-Compound 16813750± 4026065  −2 7 85 μg/kg H1H9519N-Compound 111254 ± 187288 100 7 85μg/kg H1H9521N-Compound 110413 ± 161353 100 7 85 μg/kg

TABLE 15B Inhibition of OVCAR3/luc SC Tumor Growth at Day 37 in SCIDmice treated with anti-MUC16 antibodies conjugated to Compound 7 FinalTumor Average Tumor Volume Growth Inhibition Treatment Group (mean ±SEM) (%) Vehicle 1210 ± 426 0 Control-Compound 1737 ± 391 −51 7 85 μg/kgH1H9519N-Compound  187 ± 269 100 7 85 μg/kg H1H9521N-Compound  89 ± 97109 7 85 μg/kg

TABLE 15C Inhibition of OVCAR3/luc IP Tumor Growth at Day 49 in SCIDmice treated with anti-MUC16 antibodies conjugated to Compound 10 FinalTumor Average Tumor Radiance Growth Inhibition Treatment Group (mean ±SEM) (%) Vehicle 29211000 ± 23504780 0 Control-Compound 17332625 ±14346694 41 10 85 μg/kg Control-Compound 32075000 ± 15623403 −10 10 170μg/kg Control-Compound 22882350 ± 18771913 22 10 340 μg/kgH1H9521N-Compound 574285 ± 306844 99 10 85 μg/kg H1H9521N-Compound236037 ± 226948 100 10 170 μg/kg H1H9521N-Compound 26472 ± 25079 101 10340 μg/kg

Example 11: Generation of Anti-CD3 Antibodies

Anti-CD3 antibodies were obtained by immunizing an engineered mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions with cells expressing CD3 or with DNA encoding CD3. Theantibody immune response was monitored by a CD3-specific immunoassay.When a desired immune response was achieved, splenocytes were harvestedand fused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce CD3-specific antibodies.Using this technique several anti-CD3 chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained. In addition, several fully human anti-CD3 antibodies wereisolated directly from antigen-positive B cells without fusion tomyeloma cells, as described in US 2007/0280945A1.

Certain biological properties of the exemplary anti-CD3 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples herein.

Example 12: Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 16 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-CD3 antibodies ofthe invention. The corresponding nucleic acid sequence identifiers areset forth in Table 17. Methods of making the anti-CD3 antibodiesdisclosed herein can also be found in US publication 2014/0088295.

TABLE 16 Amino Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H2712N 402 404406 408 410 412 414 416 H1M2692N 418 420 422 424 426 428 430 432H1M3542N 434 436 438 440 442 444 446 448 H1M3544N 450 452 454 456 458460 462 464 H1M3549N 466 468 470 472 474 476 478 480 H1M3613N 482 484486 488 490 492 494 496 H2M2689N 498 500 502 504 506 508 510 512H2M2690N 514 516 518 520 522 524 526 528 H2M2691N 530 532 534 536 538540 542 544 H2M2704N 546 548 550 552 554 556 558 560 H2M2705N 562 564566 568 570 572 574 576 H2M2706N 578 580 582 584 586 588 590 592H2M2707N 594 596 598 600 602 604 606 608 H2M2708N 610 612 614 616 618620 622 624 H2M2709N 626 628 630 632 634 636 638 640 H2M2710N 642 644646 648 650 652 654 656 H2M2711N 658 660 662 664 666 668 670 672H2M2774N 674 676 678 680 682 684 686 688 H2M2775N 690 692 694 696 698700 702 704 H2M2776N 706 708 710 712 714 716 718 720 H2M2777N 722 724726 728 730 732 734 736 H2M2778N 738 740 742 744 746 748 750 752H2M2779N 754 756 758 760 762 764 766 768 H2M2789N 770 772 774 776 778780 782 784 H2M2862N 786 788 790 792 794 796 798 800 H2M2885N 802 804806 808 810 812 814 816 H2M2886N 818 820 822 824 826 828 830 832H2M3540N 834 836 838 840 842 844 846 848 H2M3541N 850 852 854 856 858860 862 864 H2M3543N 866 868 870 872 874 876 878 880 H2M3547N 882 884886 888 890 892 894 896 H2M3548N 898 900 902 904 906 908 910 912H2M3563N 914 916 918 920 922 924 926 928 H1H5751P 930 932 934 936 938940 942 944 H1H5752P 946 948 950 952 954 956 958 960 H1H5753B 962 964966 968 970 972 974 976 H1H5754B 978 980 982 984 986 988 990 992H1H5755B 994 996 998 1000 1002 1004 1006 1008 H1H5756B 1010 1012 10141016 1018 1020 1022 1024 H1H5757B 1026 1028 1030 1032 1034 1036 10381040 H1H5758B 1042 1044 1046 1048 1050 1052 1054 1056 H1H5761P 1058 10601062 1064 1066 1068 1070 1072 H1H5763P 1074 1076 1078 1080 1082 10841086 1088 H1H5764P 1090 1092 1094 1096 1098 1100 1102 1104 H1H5769P 11061108 1110 1112 1114 1116 1118 1120 H1H5771P 1122 1124 1126 1128 11301132 1134 1136 H1H5772P 1138 1140 1142 1144 1146 1148 1150 1152 H1H5777P1154 1156 1158 1160 1162 1164 1166 1168 H1H5778P 1170 1172 1174 11761178 1180 1182 1184 H1H5780P 1186 1188 1190 1192 1194 1196 1198 1200H1H5781P 1202 1204 1206 1208 1210 1212 1214 1216 H1H5782P 1218 1220 12221224 1226 1228 1230 1232 H1H5785B 1234 1236 1238 1240 1242 1244 12461248 H1H5786B 1250 1252 1254 1256 1258 1260 1262 1264 H1H5788P 1266 12681270 1272 1274 1276 1278 1280 H1H5790B 1282 1284 1286 1288 1290 12921294 1296 H1H5791B 1298 1300 1302 1304 1306 1308 1310 1312 H1H5792B 13141316 1318 1320 1322 1324 1326 1328 H1H5793B 1330 1332 1334 1336 13381340 1342 1344 H1H5795B 1346 1348 1350 1352 1354 1356 1358 1360 H1H5796B1362 1364 1366 1368 1370 1372 1374 1376 H1H5797B 1378 1380 1382 13841386 1388 1390 1392 H1H5798B 1394 1396 1398 1400 1402 1404 1406 1408H1H5799P 1410 1412 1414 1416 1418 1420 1422 1424 H1H5801B 1426 1428 14301432 1434 1436 1438 1440 H1H7194B 1442 1444 1446 1448 1634 1636 16381640 H1H7195B 1450 1452 1454 1456 1634 1636 1638 1640 H1H7196B 1458 14601462 1464 1634 1636 1638 1640 H1H7198B 1466 1468 1470 1472 1634 16361638 1640 H1H7203B 1474 1476 1478 1480 1634 1636 1638 1640 H1H7204B 14821484 1486 1488 1634 1636 1638 1640 H1H7208B 1490 1492 1494 1496 16341636 1638 1640 H1H7211B 1498 1500 1502 1504 1634 1636 1638 1640 H1H7221B1506 1508 1510 1512 1634 1636 1638 1640 H1H7223B 1514 1516 1518 15201634 1636 1638 1640 H1H7226B 1522 1524 1526 1528 1634 1636 1638 1640H1H7232B 1530 1532 1534 1536 1634 1636 1638 1640 H1H7233B 1538 1540 15421544 1634 1636 1638 1640 H1H7241B 1546 1548 1550 1552 1634 1636 16381640 H1H7242B 1554 1556 1558 1560 1634 1636 1638 1640 H1H7250B 1562 15641566 1568 1634 1636 1638 1640 H1H7251B 1570 1572 1574 1576 1634 16361638 1640 H1H7254B 1578 1580 1582 1584 1634 1636 1638 1640 H1H7258B 15861588 1590 1592 1634 1636 1638 1640 H1H7269B 1594 1596 1598 1600 16341636 1638 1640 H1H7279B 1602 1604 1606 1608 1634 1636 1638 1640H1xH7221G 1610 1612 1614 1616 1634 1636 1638 1640 H1xH7221G3 1618 16201622 1624 1634 1636 1638 1640 H1xH7221G5 1626 1628 1630 1632 1634 16361638 1640

TABLE 17 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H2712N 401403 405 407 409 411 413 415 H1M2692N 417 419 421 423 425 427 429 431H1M3542N 433 435 437 439 441 443 445 447 H1M3544N 449 451 453 455 457459 461 463 H1M3549N 465 467 469 471 473 475 477 479 H1M3613N 481 483485 487 489 491 493 495 H2M2689N 497 499 501 503 505 507 509 511H2M2690N 513 515 517 519 521 523 525 527 H2M2691N 529 531 533 535 537539 541 543 H2M2704N 545 547 549 551 553 555 557 559 H2M2705N 561 563565 567 569 571 573 575 H2M2706N 577 579 581 583 585 587 589 591H2M2707N 593 595 597 599 601 603 605 607 H2M2708N 609 611 613 615 617619 621 623 H2M2709N 625 627 629 631 633 635 637 639 H2M2710N 641 643645 647 649 651 653 655 H2M2711N 657 659 661 663 665 667 669 671H2M2774N 673 675 677 679 681 683 685 687 H2M2775N 689 691 693 695 697699 701 703 H2M2776N 705 707 709 711 713 715 717 719 H2M2777N 721 723725 727 729 731 733 735 H2M2778N 737 739 741 743 745 747 749 751H2M2779N 753 755 757 759 761 763 765 767 H2M2789N 769 771 773 775 777779 781 783 H2M2862N 785 787 789 791 793 795 797 799 H2M2885N 801 803805 807 809 811 813 815 H2M2886N 817 819 821 823 825 827 829 831H2M3540N 833 835 837 839 841 843 845 847 H2M3541N 849 851 853 855 857859 861 863 H2M3543N 865 867 869 871 873 875 877 879 H2M3547N 881 883885 887 889 891 893 895 H2M3548N 897 899 901 903 905 907 909 911H2M3563N 913 915 917 919 921 923 925 927 H1H5751P 929 931 933 935 937939 941 943 H1H5752P 945 947 949 951 953 955 957 959 H1H5753B 961 963965 967 969 971 973 975 H1H5754B 977 979 981 983 985 987 989 991H1H5755B 993 995 997 999 1001 1003 1005 1007 H1H5756B 1009 1011 10131015 1017 1019 1021 1023 H1H5757B 1025 1027 1029 1031 1033 1035 10371039 H1H5758B 1041 1043 1045 1047 1049 1051 1053 1055 H1H5761P 1057 10591061 1063 1065 1067 1069 1071 H1H5763P 1073 1075 1077 1079 1081 10831085 1087 H1H5764P 1089 1091 1093 1095 1097 1099 1101 1103 H1H5769P 11051107 1109 1111 1113 1115 1117 1119 H1H5771P 1121 1123 1125 1127 11291131 1133 1135 H1H5772P 1137 1139 1141 1143 1145 1147 1149 1151 H1H5777P1153 1155 1157 1159 1161 1163 1165 1167 H1H5778P 1169 1171 1173 11751177 1179 1181 1183 H1H5780P 1185 1187 1189 1191 1193 1195 1197 1199H1H5781P 1201 1203 1205 1207 1209 1211 1213 1215 H1H5782P 1217 1219 12211223 1225 1227 1229 1231 H1H5785B 1233 1235 1237 1239 1241 1243 12451247 H1H5786B 1249 1251 1253 1255 1257 1259 1261 1263 H1H5788P 1265 12671269 1271 1273 1275 1277 1279 H1H5790B 1281 1283 1285 1287 1289 12911293 1295 H1H5791B 1297 1299 1301 1303 1305 1307 1309 1311 H1H5792B 13131315 1317 1319 1321 1323 1325 1327 H1H5793B 1329 1331 1333 1335 13371339 1341 1343 H1H5795B 1345 1347 1349 1351 1353 1355 1357 1359 H1H5796B1361 1363 1365 1367 1369 1371 1373 1375 H1H5797B 1377 1379 1381 13831385 1387 1389 1391 H1H5798B 1393 1395 1397 1399 1401 1403 1405 1407H1H5799P 1409 1411 1413 1415 1417 1419 1421 1423 H1H5801B 1425 1427 14291431 1433 1435 1437 1439 H1H7194B 1441 1443 1445 1447 1633 1635 16371639 H1H7195B 1449 1451 1453 1455 1633 1635 1637 1639 H1H7196B 1457 14591461 1463 1633 1635 1637 1639 H1H7198B 1465 1467 1469 1471 1633 16351637 1639 H1H7203B 1473 1475 1477 1479 1633 1635 1637 1639 H1H7204B 14811483 1485 1487 1633 1635 1637 1639 H1H7208B 1489 1491 1493 1495 16331635 1637 1639 H1H7211B 1497 1499 1501 1503 1633 1635 1637 1639 H1H7221B1505 1507 1509 1511 1633 1635 1637 1639 H1H7223B 1513 1515 1517 15191633 1635 1637 1639 H1H7226B 1521 1523 1525 1527 1633 1635 1637 1639H1H7232B 1529 1531 1533 1535 1633 1635 1637 1639 H1H7233B 1537 1539 15411543 1633 1635 1637 1639 H1H7241B 1545 1547 1549 1551 1633 1635 16371639 H1H7242B 1553 1555 1557 1559 1633 1635 1637 1639 H1H7250B 1561 15631565 1567 1633 1635 1637 1639 H1H7251B 1569 1571 1573 1575 1633 16351637 1639 H1H7254B 1577 1579 1581 1583 1633 1635 1637 1639 H1H7258B 15851587 1589 1591 1633 1635 1637 1639 H1H7269B 1593 1595 1597 1599 16331635 1637 1639 H1H7279B 1601 1603 1605 1607 1633 1635 1637 1639H1xH7221G 1609 1611 1613 1615 1633 1635 1637 1639 H1xH7221G3 1617 16191621 1623 1633 1635 1637 1639 H1xH7221G5 1625 1627 1629 1631 1633 16351637 1639

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “H1M,” “H2M,” etc.), followed by anumerical identifier (e.g. “2712,” “2692,” etc., as shown in Table 1),followed by a “P,” “N,” or “B” suffix. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1H2712N,” “H1M2692N,” “H2M2689N,” etc. The H1H, H1M and H2M prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H1H” antibody has ahuman IgG1 Fc, an “HIM” antibody has a mouse IgG1 Fc, and an “H2M”antibody has a mouse IgG2 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 1—will remain the same, and the bindingproperties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Tables 18 and 19 set out the amino acid sequence identifiers for heavychain variable regions (Table 18) and light chain variable regions(Table 19), and their corresponding CDRs, of additional anti-CD3 HCVRsand LCVRs useful in anti-MUC16×anti-CD3 bispecific antibodies of theinvention.

TABLE 18 (Heavy Chain Variable Region Amino Acid Sequences) Heavy ChainSEQ ID NOs Identifier HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1642 1644 16461648 CD3-VH-B 1658 1660 1662 1664 CD3-VH-C 1674 1676 1678 1680 CD3-VH-D1690 1692 1694 1696 CD3-VH-E 1706 1708 1710 1712 CD3-VH-F^(#) 1721 17221723 1724

TABLE 19 (Light Chain Variable Region Amino Acid Sequences) Light ChainSEQ ID NOs Identifier LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1650 1652 16541656 CD3-VL-B 1666 1668 1670 1672 CD3-VL-C 1682 1684 1686 1688 CD3-VL-D1698 1700 1702 1704 CD3-VL-E 1714 1716 1718 1720 CD3-VL-F^(#) 1725 17261727 1728

The heavy and light chain variable regions of CD3-VH-F and CD3-VL-F werederived from the anti-CD3 antibody designated “L2K” as set forth inWO2004/106380.

In addition, Tables 20 and 21 set out the sequence identifiers for thenucleotide sequences encoding the heavy chain variable regions (Table20) and light chain variable regions (Table 21), and their correspondingCDRs, of additional anti-CD3 HCVRs and LCVRs useful inanti-MUC16×anti-CD3 bispecific antibodies of the invention.

TABLE 20 (Nucleotide Sequences Encoding Heavy Chain Variable RegionSequences) Heavy Chain SEQ ID NOs Identifier HCVR HCDR1 HCDR2 HCDR3CD3-VH-AA 1641 1643 1645 1647 CD3-VH-B 1657 1659 1661 1663 CD3-VH-C 16731675 1677 1679 CD3-VH-D 1689 1691 1693 1695 CD3-VH-E 1705 1707 1709 1711

TABLE 21 (Nucleotide Sequences Encoding Light Chain Variable RegionSequences) Light Chain SEQ ID NOs Identifier LCVR LCDR1 LCDR2 LCDR3CD3-VL-AA 1649 1651 1653 1655 CD3-VL-B 1665 1667 1669 1671 CD3-VL-C 16811683 1685 1687 CD3-VL-D 1697 1699 1701 1703 CD3-VL-E 1713 1715 1717 1719

Control Constructs Used in the Following Examples

Various control constructs (anti-CD3 antibodies) were included in thefollowing experiments for comparative purposes: “OKT-3,” a mousemonoclonal antibody against human T-cell surface antigens available fromthe American Type Culture Collection (ATCC) under catalog no. CRL-8001;and “SP34,” a commercially available mouse monoclonal antibody obtained,e.g., from Biolegend, San Diego, Calif. (Cat. No. 302914) or BDPharmagen, Cat. 55052, reactive against the epsilon chain of the T3complex on human T lymphocyte cells.

Example 13: Generation of Additional Anti-CD3 Antibodies

The following procedures were aimed at identifying antibodies thatspecifically recognized CD3 (T cell co-receptor) as an antigen.

A pool of anti-CD3 antibodies were derived from a genetically modifiedmouse. Briefly, mice were immunized with a CD3 antigen and generated Bcells that comprised a diversity of human VH rearrangements in order toexpress a diverse repertoire of high-affinity antigen-specificantibodies. Antibodies described in Tables 22-25 have the same lightchain sequence of VK1-39JK5 (LCVR set forth in SEQ ID NO: 1890).

Generated antibodies were tested for binding to human and cynomolgusmonkey CD3 antigen in an in vitro binding assay, and e.g. one CD3antibody: designated CD3-VH-P (HCVR set forth in SEQ ID NO: 1882) wasidentified, amongst a few others, that were found to bind to both humanand cynomolgus CD3 having an EC₅₀ between 1 and 40 nM binding (or cellbinding titration), as determined in a FACS titration of Jurkat cellsand cynomolgus T cells, respectively. See also, e.g., FACS bindingexperiments outlined in Example 15 and in PCT/US2016/044732 filed Jul.29, 2016.

The germline amino acid residues of CD3-VH-P were subsequentlyidentified (V-D-J rearrangement for CD3-VH-P is IGHV3-9*01, IGHJ6*02,IGHD5-12*01) and an antibody designated “CD3-VH-G” was engineered tocontain only germline frameworks. Other antibody derivatives wereengineered by well-known molecular cloning techniques to replace aminoacid residues in a stepwise manner based on differences between thegermline sequence and the CD3-VH-P sequence. Each antibody derivative isgiven a “CD3-VH-G” number designation. See Table 18.

While CD3-VH-G and some other engineered antibodies retained theirbinding as seen in the FACS assays, several anti-CD3 antibodies bound tohuman or cynomolgus CD3 in vitro with weak to no measurable binding,such as 40 nM EC50. Binding affinities, binding kinetics, and otherbiological properties to elucidate toxicity and pharmacokinetic (pK)profiles were subsequently investigated for bispecific antibodiescomprising the exemplary anti-CD3 antibodies generated in accordancewith the methods of this Example, are described in detail in theExamples herein.

Example 14: Heavy and Light Chain Variable Regions (Amino Acid andNucleic Acid Sequences of the CDRs)

Table 22 sets forth the amino acid sequence identifiers of the heavychain variable regions and CDRs of selected anti-CD3 antibodies of theinvention. The corresponding nucleic acid sequence identifiers are setforth in Table 23.

Amino acid and nucleic acid sequences were determined for each antibodyheavy chain sequence. Each antibody heavy chain derived from thegermline sequence (SEQ ID NO: 1910) was assigned a “G” numberdesignation for consistent nomenclature. Table 22 sets forth the aminoacid sequence identifiers of the heavy chain variable regions and CDRsof engineered anti-CD3 antibodies of the invention. The correspondingnucleic acid sequence identifiers are set forth in Table 23. The aminoacid and nucleic acid sequence identifiers of the light chain variableregion and CDRs are also identified below in Tables 24 and 25,respectively.

TABLE 22 Heavy Chain Amino Acid Sequence Identifiers Antibody CD3-VH SEQID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 1730 1732 1734 1736CD3-VH-G2 1738 1740 1742 1744 CD3-VH-G3 1746 1748 1750 1752 CD3-VH-G41754 1756 1758 1760 CD3-VH-G5 1762 1764 1766 1768 CD3-VH-G8 1770 17721774 1776 CD3-VH-G9 1778 1780 1782 1784 CD3-VH-G10 1786 1788 1790 1792CD3-VH-G11 1794 1796 1798 1800 CD3-VH-G12 1802 1804 1806 1808 CD3-VH-G131810 1812 1814 1816 CD3-VH-G14 1818 1820 1822 1824 CD3-VH-G15 1826 18281830 1832 CD3-VH-G16 1834 1836 1838 1840 CD3-VH-G17 1842 1844 1846 1848CD3-VH-G18 1850 1852 1854 1856 CD3-VH-G19 1858 1860 1862 1864 CD3-VH-G201866 1868 1870 1872 CD3-VH-G21 1874 1876 1878 1880 CD3-VH-P 1882 18841886 1888

TABLE 23 Heavy Chain Nucleic Acid Sequence Identifiers Antibody CD3-VHSEQ ID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 1729 1731 1733 1735CD3-VH-G2 1737 1739 1741 1743 CD3-VH-G3 1745 1747 1749 1751 CD3-VH-G41753 1755 1757 1759 CD3-VH-G5 1761 1763 1765 1767 CD3-VH-G8 1769 17711773 1775 CD3-VH-G9 1777 1779 1781 1783 CD3-VH-G10 1785 1787 1789 1791CD3-VH-G11 1793 1795 1797 1799 CD3-VH-G12 1801 1803 1805 1807 CD3-VH-G131809 1811 1813 1815 CD3-VH-G14 1817 1819 1821 1823 CD3-VH-G15 1825 18271829 1831 CD3-VH-G16 1833 1835 1837 1839 CD3-VH-G17 1841 1843 1845 1847CD3-VH-G18 1849 1851 1853 1855 CD3-VH-G19 1857 1859 1861 1863 CD3-VH-G201865 1867 1869 1871 CD3-VH-G21 1873 1875 1877 1879 CD3-VH-P 1881 18831885 1887

TABLE 24 Light Chain Amino Acid Sequence Identifiers Antibody SEQ IDNOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 1890 1892 1894 1896

TABLE 25 Light Chain Nucleic Acid Sequence Identifiers Antibody SEQ IDNOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 1889 1891 1893 1895

Control 1 antibody designated “CD3-L2K” was constructed based on a knownanti-CD3 antibody (i.e., the anti-CD3 antibody “L2K” as set forth inWO2004/106380).

Isotype Control Antibody, referred to in the Examples herein, is anisotype matched (modified IgG4) antibody that interacts with anirrelevant antigen, i.e. FelD1 antigen.

Example 15: In Vitro and In Vivo Studies on Human Monoclonal Anti-CD3Antibodies

In vivo and in vitro studies on human monoclonal anti-CD3 antibodieswere done as described in US publication 2014/0088295 published Mar. 27,2014, and PCT/US2016/044732 filed Jul. 29, 2016, which are herebyincorporated by reference.

Some human monoclonal anti-CD3 antibodies of the present invention bindsoluble heterodimeric CD3 protein, in either antibody-capture orantigen-capture formats, with high affinity. Soluble heterodimeric CD3protein (hCD3-epsilon/hCD3-delta; SEQ ID NOs:1900/1901) was preparedwith either a human Fc tag (hFcΔAdp/hFc; SEQ ID NOs:1931/1932) or amouse Fc tag (mFcΔAdp/mFc; SEQ ID NOs:1933/1934). Heterodimeric CD3protein was purified using the method described in Davis et al.(US2010/0331527).

Some human monoclonal anti-CD3 antibodies of the invention bound humanT-cells and induced T-cell proliferation. Some human monoclonal anti-CD3antibodies of the invention bound CD2+CD4+ monkey T-cells and inducedtheir proliferation. Some human monoclonal anti-CD3 antibodies supportedredirected T-cell mediated killing via Fc/FcR interaction in a calceinbased U937 killing assay. The observed killing, believed to be dependenton the antibody's Fc engagement with the Fc Receptor on U937 cellsleading to clustering of CD3 on adjacent T-cells, was squelched byaddition of non-specific human IgG (data not shown). A wide range ofbispecific antibodies constructed with anti-CD3 arm variants describedherein (particularly anti-CD3 arms based on the CD3-VH-P heavy chainderived from IGHV3-9*01, IGHJ6*02, IGHD5-12*01) activate human PBMCcells, and monkey PBMCs, and display cytotoxic activity on tumorantigen-expressing cell lines.

Example 16: Screening and Identification of Anti-MUC16 MonoclonalAntibodies Suitable for Immunohistochemistry (IHC) on Formalin FixedParaffin Embedded (FFPE) Samples

Human tumor cell lines with known levels of expression of MUC16 wereidentified, fixed in 10% Neutral Buffered Formalin and embedded inparaffin. These lines were used to screen various anti-MUC16 antibodiesto identify candidates for IHC studies.

Cell lines included the following MUC16 Negative cell lines:

HT29 (Colon), and PC3/ATCC parental (Prostate); Pancreatic cell lineswith low to no levels of MUC16: Capan1 (Pancreatic adenocarcinoma), HPAC(Pancreatic adenocarcinoma). Endogenously MUC16-expressing cell linesinclude: OVCAR3 (Ovarian) and PEO-1 (Serous ovarian carcinoma).

Transfected cell lines were engineered as follows: PC3/ATCC (parentalProstate cancer line) cells were transfected to generate PC3/MUC16“short” and PC3/MUC16 “high” cell lines. Both constructs include theC-terminal domain of MUC16 from amino acids 13,810-14,507 (of SEQ ID NO:1899), and this includes part of the SEA12 domain, SEA13, SEA14, SEA15,SEA16, the C-terminal non-SEA region, the transmembrane region and thecytoplasmic domain. In addition, the PC3/MUC high cells have additionalN-terminal amino acids 12783-13467 (of SEQ ID NO: 1899) which includeSEA5 (partial) through SEA9 and a short linker between the SEA9 domainand the start of the MUC16 short construct. This allows fordifferentiation between anti-MUC16 antibodies that bind in the repeatregion and those that bind to the “nub” portion of MUC16 adjacent to themembrane following enzymatic cleavage and release of the repeat regions(analogous to the CA125 portion of MUC16).

All staining was performed on the Ventana Discovery XT autostainer usingstandard protocols. Cell pellets were deparaffinized, Heat InducedEpitope Retrieval was optimized, endogenous Biotin was blocked andProtein blocking was performed. The antibodies were applied manually atan initial concentration of 10 μg/ml and were also titrated down toensure the specificity of the signal. Comparison was made against acommercially available anti-MUC16 antibody (OC-125), which is specificfor the repeat regions of CA-125 (Roche, Ventana Catalog #760-2610), anda negative control (absence of primary antibody). Detection was withDonkey anti-mouse-Biotin followed by Streptavidin-Horseradishperoxidase. The conversion of the substrate, Di-amino Benzidine (DAB)was observed as brown staining. Samples were counterstained withhematoxylin to visualize the nuclei. The results of the stainingexperiments are presented in Table 26, below.

TABLE 26 Binding of anti-MUC16 Antibodies to MUC16-negative Cells, andCells Expressing MUC16 or Membrane-Proximal Portions of MUC16 PC3/ATCCPC3/MUC16 PC3/MUC16 Cell line: HT29 Capan1 HPAC OVCAR3 PEO-1 parentalShort High H1M7130N − − − +++ ++ − +++ +++ H2aM7128N − − + +++ ++ − ++++++ H2aM7131N − − − +++ ++ − +++ +++ H2aM7133N − − − +++ ++ − − −H2aM7138N − − + +++ ++ − +++ +++ H1M9519N − NT NT +++ +++ NT − +++H3M9525N − NT NT − − NT − − OC-125 − − + +++ ++ − − +++ Neg. Control − −− − − − − − NT—not tested

Four of the tested antibodies (H1M7130N, H2aM7128N, H2aM7131N andH2aM7138N) showed positive binding to cells expressing the “nub” portionof MUC16 without the repeat regions (PC3/MUC16 short). These antibodiesare identified as “nub binders.” One of the tested antibodies (H1M9519N)showed positive binding to cells expressing the repeat regions of MUC16(PC3/MUC16 high), but negative binding to cells expressing only the“nub” portion of MUC16 (PC3/MUC16 short). This antibody is identified asa “repeat binder,” similar to the commercially available OC-125antibody. It is expected that the tested antibodies bind tissues orcells of different origin having the expressed proteins and/or proteinfragments as described herein.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

TABLE 27 Sequences Excluded from ST.26-Formatted Sequence ListingSEQ ID NO: Sequence 13 gctgcatcc 14 AAS 29 actgcatcc 30 TAS 45 actgcatcc46 TAS 61 actgcatcc 62 TAS 77 gctgcatcc 78 AAS 93 Gctgcatcc 94 AAS 109ggtgcatcc 110 GAS 125 ggtgcatcc 126 GAS 141 ggtatatcc 142 GIS 157ggtgcatcc 158 GAS 173 ggtgcatcc 174 GAS 189 ggtgcatcc 190 GAS 213gatgcatcc 214 DAS 229 ggtgcatcc 230 GAS 245 ggtgcatcc 246 GAS 269ggtgcatcc 270 GAS 293 ggtgcatcc 294 GAS 309 gctgcatcc 310 AAS 325gctgcatcc 326 AAS 341 ggtgcatcc 342 GAS 357 tgggcatct 358 WAS 373tgggcatct 374 WAS 389 tgggcatct 390 WAS 397 gctgcatcc 398 AAS 413ggtgcatcc 414 GAS 429 ggtgcatcc 430 GAS 445 gctgcatcc 446 AAS 461ggtgcatcc 462 GAS 477 gctgcatcc 478 AAS 493 gctgcatcc 494 AAS 509gatgcatcc 510 DAS 525 ggtgcatcc 526 GAS 541 ggtgcatcc 542 GAS 557ggtgcatcc 558 GAS 573 ggtgcatcc 574 GAS 589 ggtgcatcc 590 GAS 605ggtgcatcc 606 GAS 621 ggtgcatcc 622 GAS 637 ggtgcatcc 638 GAS 653ggtgcatcc 654 GAS 669 ggtgcatcc 670 GAS 685 ggtgcatcc 686 GAS 701ggtgcaacc 702 GAT 717 ggtgcaacc 718 GAT 733 ggtgcatcc 734 GAS 749ggtgcatcc 750 GAS 765 tgggcatct 766 WAS 781 gctgcatcc 782 AAS 797ggtgcatcc 798 GAS 813 ggtgcaacc 814 GAT 829 ggtgcaacc 830 GAT 845gctgcatcc 846 AAS 861 gctgcatcc 862 AAS 877 gctgcatcc 878 AAS 893gctgcatcc 894 AAS 909 gctgcatcc 910 AAS 925 gctgcatcc 926 AAS 941gctgcttcc 942 AAS 957 gaagcttct 958 EAS 973 gctgtatcc 974 AVS 989gatgcatcc 990 DAS 1005 gctgcatcc 1006 AAS 1021 gctgcatcc 1022 AAS 1037gctgcatcc 1038 AAS 1053 gctgcatcc 1054 AAS 1069 gctgcatcc 1070 AAS 1085gctgcgtcc 1086 AAS 1101 gctgcatcc 1102 AAS 1117 ggtgcgtcc 1118 GAS 1133ggtgcgtcc 1134 GAS 1149 ggtgcgtcc 1150 GAS 1165 ggtgcgtcc 1166 GAS 1181ggtgcatcc 1182 GAS 1197 ggtgcgtcc 1198 GAS 1213 ggtgcgtcc 1214 GAS 1229aaggcgtct 1230 KAS 1245 tgggcatct 1246 WAS 1261 actgcatcc 1262 TAS 1277ggtgcatcc 1278 GAS 1293 actgcatcc 1294 TAS 1309 actgcatcc 1310 TAS 1325actgcatcc 1326 TAS 1341 gttgcatcc 1342 VAS 1357 actgcatcc 1358 TAS 1373gctgcatcc 1374 AAS 1389 gttgcatcc 1390 VAS 1405 actgcatcc 1406 TAS 1421actgcatcc 1422 TAS 1437 gttgcatcc 1438 VAS 1637 gctgcatcc 1638 AAS 1653ggtgcatcc 1654 GAS 1669 ggtgcatcc 1670 GAS 1685 ggtgcatcc 1686 GAS 1701ggtgcatcc 1702 GAS 1717 ggtgcatcc 1718 GAS 1727 DTS 1893 actgcatcc 1894TAS 1939 ggtgcatcc 1940 GAS 1955 ggtgcatcc 1956 GAS

1-71. (canceled)
 72. A group of nucleic acid molecules encoding abispecific antigen-binding molecule that binds human CD3 and humanMUC16, wherein the group of nucleic acid molecules comprises: (a) afirst nucleic acid molecule encoding a heavy chain variable region(HCVR) comprising HCDR1, HCDR2, HCDR3 domains, respectively, comprisingthe amino acid sequences of SEQ ID NOs: 1732, 1734 and 1736; (b) asecond nucleic acid molecule encoding a HCVR comprising HCDR1, HCDR2,HCDR3 domains, respectively, comprising the amino acid sequences of SEQID NOs: 20, 22 and 24; and (c) a third nucleic acid molecule encoding alight chain variable region (LCVR) comprising LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 28, 30 and
 32. 73. The group of nucleic acid molecules of claim 72,wherein: (a) the first nucleic acid molecule encodes a HCVR comprisingthe amino acid sequence of SEQ ID NO: 1730; (b) the second nucleic acidmolecule encodes a HCVR comprising the amino acid sequence of SEQ ID NO:18; and (c) the third nucleic acid molecule encodes a LCVR comprisingthe amino acid sequence of SEQ ID NO:
 26. 74. The group of nucleic acidmolecules of claim 72, wherein the bispecific antigen-binding moleculeis a bispecific antibody, the first nucleic acid molecule encodes afirst heavy, the second nucleic acid molecule encodes a second heavychain, and the third nucleic acid molecule encodes a light chain. 75.The group of nucleic acid molecules of claim 74, wherein the first heavychain or the second heavy chain, but not both, comprises a CH3 domaincomprising a H435R (EU numbering) modification and a Y436F (EUnumbering) modification.
 76. The group of nucleic acid molecules ofclaim 74, wherein the first heavy chain, the second heavy chain, or boththe first and second heavy chains comprise a human IgG1 heavy chainconstant region, or a human IgG4 heavy chain constant region.
 77. Thegroup of nucleic acid molecules of claim 74, wherein: (a) the firstnucleic acid molecule encodes a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 1961; (b) a second nucleic acid moleculeencoding a second heavy chain comprising the amino acid sequence of SEQID NO: 1959; and (c) a third nucleic acid molecule encoding a lightchain comprising the amino acid sequence of SEQ ID NO:
 1960. 78. Thegroup of nucleic acid molecules of claim 73, wherein the first nucleicacid molecule comprises the nucleotide sequence of SEQ ID NO: 1729, thesecond nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 17, and the third nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO:
 25. 79. A group of nucleic acid moleculesencoding a bispecific antigen-binding molecule that binds human CD3 andhuman MUC16, wherein the group of nucleic acid molecules comprises: (a)a first nucleic acid molecule encoding a heavy chain variable region(HCVR) comprising HCDR1, HCDR2, HCDR3 domains, respectively, comprisingthe amino acid sequences of SEQ ID NOs: 1868, 1870 and 1872; (b) asecond nucleic acid molecule encoding a HCVR comprising HCDR1, HCDR2,HCDR3 domains, respectively, comprising the amino acid sequences of SEQID NOs: 20, 22 and 24; and (c) a third nucleic acid molecule encoding alight chain variable region (LCVR) comprising LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 28, 30 and
 32. 80. The group of nucleic acid molecules of claim 72,wherein: (a) the first nucleic acid molecule encodes a HCVR comprisingthe amino acid sequence of SEQ ID NO: 1866; (b) the second nucleic acidmolecule encodes a HCVR comprising the amino acid sequence of SEQ ID NO:18; and (c) the third nucleic acid molecule encodes a LCVR comprisingthe amino acid sequence of SEQ ID NO:
 26. 81. The group of nucleic acidmolecules of claim 79, wherein the bispecific antigen-binding moleculeis a bispecific antibody, the first nucleic acid molecule encodes afirst heavy, the second nucleic acid molecule encodes a second heavychain, and the third nucleic acid molecule encodes a light chain. 82.The group of nucleic acid molecules of claim 81, wherein the first heavychain or the second heavy chain, but not both, comprises a CH3 domaincomprising a H435R (EU numbering) modification and a Y436F (EUnumbering) modification.
 83. The group of nucleic acid molecules ofclaim 81, wherein the first heavy chain, the second heavy chain, or boththe first and second heavy chains comprise a human IgG1 heavy chainconstant region, or a human IgG4 heavy chain constant region.
 84. Thegroup of nucleic acid molecules of claim 81, wherein: (a) the firstnucleic acid molecule encodes a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 1962; (b) a second nucleic acid moleculeencoding a second heavy chain comprising the amino acid sequence of SEQID NO: 1959; and (c) a third nucleic acid molecule encoding a lightchain comprising the amino acid sequence of SEQ ID NO:
 1960. 85. Thegroup of nucleic acid molecules of claim 73, wherein the first nucleicacid molecule comprises the nucleotide sequence of SEQ ID NO: 1865, thesecond nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 17, and the third nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO:
 25. 86. An expression vector or a group ofexpression vectors comprising the group of nucleic acid molecules ofclaim
 72. 87. An expression vector or a group of expression vectorscomprising the group of nucleic acid molecules of claim
 79. 88. Anisolated host cell comprising the expression vector or the group ofexpression vectors of claim
 86. 89. An isolated host cell comprising theexpression vector or the group of expression vectors of claim
 87. 90. Amethod of producing a bispecific antigen-binding molecule that bindshuman CD3 and human MUC16, comprising culturing the host cell of claim88 under conditions permitting production of the bispecificantigen-binding molecule, and recovering the bispecific antigen-bindingmolecule so produced.
 91. The method of claim 90, further comprisingformulating the bispecific antigen-binding molecule as a pharmaceuticalcomposition with a suitable carrier.